Proinsulin C-peptide has been shown to stimulate the activity of Na+ K+ ATPase of rat renal tubule segments. Thirty-six peptides and amino acids, corresponding to parts of the intact rat C-peptide and suitable controls were screened for capacity to stimulate Na+, K+-ATPase in an attempt to determine potential active sites in the C-peptide molecule. The carboxy-terminal tetra and penta peptides were found to elicit 92-103% of the intact molecule's activity, and the remaining segment, des-(27-31) C-peptide, did not possess stimulatory activity. Peptides from the middle C-peptide segment, however, centering around a GGPEAG sequence, stimulated Na+, K+-ATPase activity (36-80% of the intact molecule's effect) but this effect was not balanced by corresponding inactivity of other parts. Furthermore, it was paralleled by activity of a non-native dipeptide D-form. It is concluded that the latter effect and that of the middle segment may represent complex interactions other than the apparently specific effects of the C-terminal segment.