OBJECTIVE:: To determine whether trigonelline contributes to the effect of coffee on homocysteine (Hcy). DESIGN AND INTERVENTIONS:: This was a randomised crossover study. Subjects consumed 50 mg trigonelline, 5 g of instant coffee (≈50 mg trigonelline) or water, consumed as a single dose in 100 ml, with 1 week between each treatment. Blood samples were drawn fasting and hourly for 8 h. Urine samples were collected pretreatment and every 2 h for 8 h. SETTING:: Christchurch Clinical Studies Trust, Christchurch, New Zealand. SUBJECTS:: Eight healthy male subjects. RESULTS:: Instant coffee raised plasma Hcy concentrations compared with water (P=0.019) and trigonelline (P=0.037). Plasma Hcy concentrations were not different between water and trigonelline treatments (P=0.789). The change in plasma Hcy concentration was higher (mean±s.e.) 4 h (0.7±0.2 μmol/l, P=0.006), 5 h (0.7±0.2 μmol/l, P=0.013) and 7 h (0.7±0.2 μmol/l, P=0.024) following coffee consumption. Urinary glycine betaine excretion was increased by coffee but not by trigonelline. CONCLUSION:: Ingestion of instant coffee acutely elevated plasma Hcy; however, trigonelline is not responsible for this rise. SPONSORSHIP:: Supported by the Health Research Council, the Canterbury Medical Foundation, the Foundation of Research, Science and Technology.