Efficient uncoating requires not only an optimal cellular environment, but also some intrinsic properties of the viral capsid protein itself. Using an in vitro uncoating model, we demonstrated that substitution of each serine residue with alanine at the three major phosphorylation sites of HIV-1 capsid protein, i.e. Ser-109, Ser-149 and Ser-178, could significantly reduce uncoating activity of purified core particles. We also showed that the core stability of mutant viruses was lower than that of the wild-type virus so that the lack of efficient uncoating of each mutant could not be due to an increase in capsid physical stability. However, serine-to-aspartic acid mutation to mimic the negative charge of phosphor-serine could not restore either uncoating activity or infectivity, and treatment of purified core particles with a phosphatase did not alter the uncoating activity. Our data indicated that mutations at phosphoacceptor sites of capsid disturbed the uncoating mechanism, but the defect may not be directly caused by the lack of phosphate on the core particles undergoing uncoating.