Dynamic in vivo imaging of spermatozoa radiolabeled with 99mTc-HMPAO in the female genital tract was performed noninvasively. Animal studies were performed under a variety of insemination conditions and their influence on the dynamics and number of spermatozoa in ascent was demonstrated. Spermatozoa motility was not significantly reduced by radiolabeling. In vitro and in vivo labeling stability was > 93% after 18 hr. Female rabbits were inseminated with labeled spermatozoa and dynamic scintigraphic studies were performed up to 12 hr after insemination. The in vivo behavior of labeled spermatozoa was similar to native spermatozoa. Absolute spermatozoa numbers could be determined noninvasively and reliably at different parts in the female genital tract at various timepoints after insemination.