The effects of age and gonads were studied in rats subjected to open-field tests, during which ambulation behavior was recorded. Subjects were three groups of male and female rats: sham-operation on day 1 and day 21; gonadectomy on day 1 and sham-operation on day 21; and sham-operation on day 1 and gonadectomy on day 21. Half of each group were tested in a circular open field (3 min/day, 3 consecutive days) on days 28-30; the others were tested on days 47-49. Representatives of both batches were tested again in a square open field on days 76-78. There was a sex difference in ambulation at 77 days, but not at earlier ages. In animals gonadectomized on day 1 or day 21 the sex difference in adulthood failed to occur, because castration caused the males to ambulate as much as sham-operated and ovariectomized females. On the basis of our results and reports in the literature it is suggested that testicular secretions around puberty have an organizing effect on ambulation behavior. The intact adult male rat ambulates less than the adult female and this difference persists after castration in adulthood. Castration well before puberty prevents the development of the adult sex difference.