This paper presents the progress made in optimizing the effect of the presence or absence of corpus luteum (CL) on the follicular population during superovulation in Murrah buffaloes. The animals were divided into two groups as follows: (1) CL group (n = 10) received a total dose of 75 NIH units of follicle stimulating hormone (FSH) in a descending dose schedule spread over the first 3 days (twice daily) after spontaneous ovulation (day 0); (2) Non-CL group (n = 10) received prostaglandin F2α at 11 days after ovulation. After 36 h, all the follicles (larger than 5 mm) were aspirated (day 0). The FSH treatment started 24 hr after aspiration and continued for 3 days, similar to CL group. The number of small (5 to <8mm), medium (8 to <10mm), and large (≥10 mm) follicles was examined on days 1, 3, and 5 in all groups. Blood samples were collected daily for 5 days, progesterone (P4), and estradiol (E2) in plasma were measured by radioimmunoassay. The results showed that in CL group, the P4 level increased gradually from 0.75 ng/ml at day 1 to 2.5 ng/ml at day 5, whereas in the non-CL group, the P4 level was completely below 0.75 ng/ml. All buffaloes of the non-CL group showed an increase of E2 at day 3 or day 5. Moreover, at the end of the treatment, the number of follicles in the non-CL group was significantly increased compared with that of the CL group (22.8 ± 2.0 vs. 11.6 ± 2.0). In conclusion, enhanced superovulation and E2 secretion during FSH treatment were observed. Therefore, the absence of the CL may play a critical role in the superovulation response by controlling the number of growing follicles.