Properties of human profilin I mutated in the major actin-binding site were studied and compared with wild-type profilin using beta/gamma-actin as interaction partner. The mutants ranged in affinity, from those that only weakly affected polymerization of actin to one that bound actin more strongly than wild-type profilin. With profilins, whose sequestering activity was low, the concentration of free actin monomers observed at steady-state of polymerization [Afree], was close to that seen with actin alone ([Acc], critical concentration of polymerization). Profilin mutants binding actin with an intermediate affinity like wild-type profilin caused a lowering of [Afree] as compared to [Acc], indicating that actin monomers and profilin:actin complexes participate in polymer formation. With a mutant profilin, which bound actin more strongly than the wild-type protein, an efficient sequestration of actin was observed, and in this case, the [Afree] at steady state was again close to [Acc], suggesting that the mutant profilin:actin had a greatly lowered ability to incorporate actin subunits at the (+)-end. The results from the kinetic and steady-state experiments presented are consonant with the idea that profilin:actin complexes are directly incorporated at the (+)-end of actively polymerizing actin filaments, while they do not support the view that profilin facilitates polymer formation.