During the process of capacitation, spermatozoa undergo significant changes in membrane composition, including removal of decapacitating factors (DFs), which are present in seminal plasma, that lead to increased sensitivity to physiological stimuli of the acrosome reaction. In the present study we investigated the presence, localization, and effects on human spermatozoa of 2 proteins of seminal plasma origin, uteroglobin (UG) and trans‐glutaminase (TG). These 2 proteins interact with one another because TG promotes covalent links of UG to sperm surface proteins. We found that UG is localized around the entire surface of ejaculated human sperm, whereas TG is predominantly localized in the neck. FACScan analysis confirmed the surface localization of both antigens and demonstrated that swim‐up selection of spermatozoa was associated with a significant reduction in the contents of the 2 substances when compared with unselected samples. Western blot analysis of UG in total sperm lysates confirmed the lower content of the protein in swim‐up—selected sperm. Swim‐up—selected sperm were characterized by their ability to undergo a spontaneous, time‐dependent increase of capacitation‐characteristic chlortetracycline pattern of fluorescence and increase in responsiveness to progesterone. Such changes were not observed in unselected sperm. Exogenous addition of TG, together with recombinant rabbit UG, prevented the spontaneous increase in responsiveness to progesterone (acrosome reaction and intracellular calcium) at 24 hours in swim‐up—selected sperm, suggesting the occurrence of a capacitation‐inhibiting activity of the 2 substances. In addition, we found that endogenous UG and TG contents, as determined by FACScan analysis, were negatively correlated (P < .0001) with sperm motility and that exogenous addition of the 2 substances resulted in a substantial reduction of progressive motility (P < .01). Collectively, these data indicate that TG and UG represent 2 DFs, and contribute to understanding the biochemical mechanisms that characterize the process of capacitation.