This study was performed in order to test the hypothesis that the connecting peptide of proinsulin, C-peptide, might in itself possess biological activity. Renal tubular Na⁺, K⁺-ATPase, which is a well-established target for many peptide hormones, was chosen as a model. Rat C-peptide (I) was found to stimulate Na⁺, K⁺-ATPase activity in single, proximal convoluted tubules dissected from rat kidneys. C-peptide increased the Na⁺ affinity of the enzyme and all subsequent studies were performed at non-saturating Na⁺ concentrations. C-peptide stimulation of Na⁺, K⁺-ATPase activity occurred in a concentration-dependent manner in the dose range 10^–8–10^–6 mol/l. The presence of neuropeptide Y, 5×10^–9 mol/l, enhanced this effect and stimulation of Na⁺, K⁺-ATPase activity then occurred in the C-peptide dose range 10^–11–10^–8 mol/l. C-peptide stimulation of Na⁺, K⁺-ATPase activity was abolished in tubules pretreated with pertussis toxin. It was also abolished in the presence of FK 506, a specific inhibitor of the Ca²⁺-calmodulin-dependent protein phosphatase 2B. These results indicate that C-peptide stimulates Na⁺, K⁺-ATPase activity, probably by activating a receptor coupled to a pertussis toxin-sensitive G-protein with subsequent activation of Ca²⁺-dependent intracellular signalling pathways.