Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and bacteraemia and is capable of remarkable phenotypic plasticity, responding rapidly to environmental change. Pneumococcus is a nasopharyngeal commensal, but is responsible for severe, acute infections following dissemination within-host. Pneumococcus is adept at utilising host resources, but the airways are compartmentalised and those resources are not evenly distributed. Challenges and opportunities in metabolite acquisition within different airway niches may contribute to the commensal-pathogen switch when pneumococcus moves from nasopharynx into lungs. We used NMR to characterise the metabolic landscape of the mouse airways, in health and during infection. Using paired nasopharynx and lung samples from naïve animals, we identified fundamental differences in metabolite bioavailability between airway niches. Pneumococcal pneumonia was associated with rapid and dramatic shifts in the lung metabolic environment, whilst nasopharyngeal carriage led to only modest change in upper airway metabolite profiles. NMR spectra derived from the nasopharynx of mice infected with closely-related pneumococcal strains that differ in their colonisation potential could be distinguished from one another using multivariate dimensionality reduction methods. The resulting models highlighted that increased branched-chain amino acid (BCAA) bioavailability in nasopharynx is a feature of infection with the high colonisation potential strain. Subsequent analysis revealed increased expression of BCAA transport genes and increased intracellular concentrations of BCAA in that same strain. Movement from upper to lower airway environments is associated with shifting challenges in metabolic resource allocation for pneumococci. Efficient biosynthesis, liberation or acquisition of BCAA is a feature of adaptation to nasopharyngeal colonisation. Author summary: Several species of bacterial pathogens that cause disease in humans are often found residing in the upper reaches of the airways. Within this environment, they rarely cause ill health, but they can be responsible for severe disease when they reach the lower regions of the respiratory tract, such as the lungs. The reasons why the same bacteria can be benign in one airway region and pathogenic in another are poorly understood. In this study, we defined the chemical environment of different airway regions, to determine what resources were available for bacteria to use for metabolism and growth. We identified gradients of chemical abundance within the airways, including in a group of amino acids (the building blocks of proteins) that trigger important changes in bacteria that influence their disease-causing potential. Using a pathogenic bacterial species–Streptococcus pneumoniae–we show that the ability of the bacteria to produce or acquire those same amino acids determines how successfully they can colonise the airways. Defining airway environments, and the challenges and opportunities posed to bacteria that colonise them, will help us understand the relationship between asymptomatic airway infection and diseases such as pneumonia. [ABSTRACT FROM AUTHOR]