Previous study of the development of the levator ani muscle (LA) revealed that the sex difference in muscle fiber number is under the control of testosterone during the perinatal period. In this study, the development of the LA muscle was examined in both sexes to test the hypothesis that testosterone action determines a further sex difference at the onset of puberty. This period is of key importance because plasma testosterone concentration rises sharply between Postnatal Days 40 and 60 in the male, while in the female testosterone level stays very low. In the male, a transient increase in the satellite cell number occurs between Days 42 and 47, followed by a persistent myonuclei increase. Hence, when the pubertal process has been completed, the myonuclei number is increased by about 50%. Moreover, sexual dimorphism related to the muscle fiber width becomes more marked: before puberty (Day 30), the mean cross-sectional area of muscle fibers is 2-fold higher in males than in females, while between Days 30 and 78, a further hypertrophy is observed which leads to a mean cross-sectional area about 10-fold higher in the male. In the female, during the same period, there is neither satellite cell increase nor myonuclei increase and the fiber cross-sectional area remains constant. Treatment with testosterone before the onset of puberty induces a marked but transient satellite cell increase not only in male but also in female LA muscle as early as 3 days after the time of injection. This cell proliferation is followed by a subsequent increase in the myonuclei number. Results indicate that (1) the levator ani muscle in the male exhibits high sensitivity to testosterone during the pubertal process; during this period, sexual dimorphism related to fiber width and myonuclei number is emphasized; (2) the myonuclei increase is subsequent to the testosterone-induced satellite cell proliferation; and (3) the female LA muscle presents the same sensitivity to testosterone as the male. Hence, the female muscle does not acquire specific male characteristics, owing to the very low concentration of circulating androgens.