The relaxin-like factor (RLF) is a circulating hormone that is synthesized in the gonads of mammals and released into the bloodstream. The distribution of its receptor and a trace of cross-reactivity to relaxin receptors implied that this relatively new factor is more relaxin- than insulin-like. The chemical synthesis of RLF analogues with specific modifications in positions B27 and B25, or the truncated form des(B27-31)RLF, clearly indicate that the intact indole ring in position B27 is crucial for high RLF receptor-binding. Receptor-binding was reduced by 2 orders of magnitude for Leu(B27)RLF (3%), Ala(B27)RLF (2.1%), and des(B27-31)RLF (0.4%), whereas slightly better binding was observed for His(B27)RLF (7.5%), Phe(B27)RLF (21%), D-Trp(B27) (26%), and the oxindole(B27)RLF (41%). On the basis of these observation it seems that an aromatic ring system in the beta- or gamma-position is required for binding. Structure prediction of the C-terminal region of the B chain indicated a possible type I or type III turn for residues C-G-G-P-R (B22-26) preceding the tryptophan. Exchanging Pro(B25) for D-Pro within the turn caused a severe structural rearrangement at the C terminus and a 96% drop in activity. It appears that the steric effect of L-Pro(B25) is important for the proper positioning of Trp(B27).