A simple procedure for the isolation of profilin mutants having a reduced capacity to bind poly(l-proline) is used to isolate two mutants of human profilin I, W3N and H133S. Binding of the mutants to poly(l-proline), actin, and phosphatidylinositol (4,5)-bisphosphate (PIP2) was studied. Both mutations abolished the poly(l-proline)-binding activity of profilin. This suggests that the arrangement of the N- and C-terminal helices forming the poly(l-proline)-binding site depends on the stabilizing interaction between W3 and W31 in the underlying β-strand, and that the H133S mutation in the C-terminal helix also must have distorted the arrangement of the terminal helices. Both mutations caused a reduced affinity for actin, with the W3N replacement having the most pronounced effect. This shows that structural changes in the poly(l-proline)-binding region of profilin can affect the distantly located actin-binding site. Thus, ligands influencing the structure of the poly(l-proline)-binding site may regulate actin polymerization through profilin. This is consonant with the finding that PIP2, which changes the tryptophan fluorescence in wild-type profilin and dissociates the profilin:actin complex in vitro, binds more strongly to the W3N mutant profilin. Thus, the poly(l-proline)-binding surface represents a crucial regulatory site of profilin function.