Pregnant mare serum gonadotropin (PMSG) is a long acting luteotropin with follitropin activity. Cattle were used to examine whether a smaller dose of PMSG has substantial luteotropic effect without excessive follitropic effects. Eleven Japanese Black heifers were randomly assigned to two groups. Animals were administered 500 IU PMSG on the day of ovulation (Day 0) to promote the formation of corpus luteum (group A; N = 5) or Day 7 to stimulate the luteal function (group B; N = 6). Four of them were given injections of saline on Days 0 and 7 of the preceding estrous cycle for control. All animals were examined by palpation per rectum every other morning and bled every day for steroids analyses. The length of estrous cycle was shortened by the treatment in group A compared with the previous cycle, whereas it was extended in group B (P < 0.05). Progesterone secretion was not enhanced in group A, but it was significantly elevated and sustained on higher levels in group B (P < 0.05) as compared with the control. Although estradiol-17 beta concentrations were significantly increased in both PMSG-treated groups (P < 0.05), no excessive follicular development was observed. It is concluded that 500 IU PMSG administered on Day 7 enhances luteal function without excessive follicular development, whereas the administration on Day 0 has an adverse effect on luteal function.