Extracellular K+ concentration ([K+]) is closely regulated by the concerted regulatory responses of kidney and muscle. In this study, we aimed to define the responses activated when dietary K+ was moderately reduced from a control diet (1.0% K+) to a 0.33% K+ diet for 15 days. Although body weight and baseline plasma [K+] (4.0 mM) were not reduced in the 0.33% K+ group, regulatory responses to conserve plasma [K+] were evident in both muscle and kidney. Insulin-stimulated clearance of K+ from the plasma was estimated in vivo in conscious rats with the use of tail venous and arterial cannulas. During infusion of insulin · (50 mU·kg-1·min-1), plasma [K+] level fell to 3.2 ± 0.1 mM in the 1.0% K+ diet group and to only 3.47 ± 0.07 mM in the 0.33% K+ diet group (P < 0.01) with no reduction in urinary K+ excretion, which is evidence of insulin resistance to cellular K+ uptake. Insulin-stimulated cellular K+ uptake was quantitated by measuring the K+ infusion rate necessary to clamp plasma K+ at baseline (in μmol·kg·min-1) during 5 mU of insulin·kg-1min-1 infusion: 9.7 ± 1.5 in 1% K+ diet was blunted to 5.2 ± 1.7 in the 0.33% K+ diet group (P < 0.001). Muscle [K+] and Na+-K+-ATPase activity and abundance were unchanged during the 0.33% K+ diet. Renal excretion, which was measured overnight in metabolic cages, was reduced by 80%, from 117.6 ± 10.5 μmol/h/animal (1% K+ diet) to 24.2 ± 1.7 μmol/h/animal (0.33% K+ diet) (P < 0.001). There was no significant change in total abundance of key renal K+ transporters, but 50% increases in both renal PTK cSrc abundance and ROMK phosphorylation in the 0.33% K+ vs. 1% K+ diet group, previously established to be associated with internalization of ROMK. These results indicate that plasma [K+] can be maintained during modest K+ restriction due to a decrease in insulin-stimulated cellular K+ uptake as well as renal K+ conservation mediated by inactivation of ROMK, both without a detectable change in plasma [K+]. The error signals inciting and maintaining these responses remain to be identified. [ABSTRACT FROM AUTHOR]