The effects of chicken luteinizing hormone (cLH: IRC-2 and PRC AE1-1), turkey LH (B221B and HS-5-18), bovine follicle-stimulating hormone (bFSH: HS-2-17), chicken FSH (cFSH: PRC DC3(2) and AGCQSQ113445C), and turkey FSH (B150A and HS-1-153) on steroid output were evaluated by in vitro incubation of various ovarian tissues with the gonadotrophins. Output of androstenedione and estradiol was determined by 3-hr incubations of individual whole small follicles, classified by size and color as follows: small white (SWF, less than 1 mm), large white (LWF, 2-3 mm), and small yellow follicles (SYF, 5-10 mm). The effects of gonadotrophin preparations were also evaluated in large preovulatory follicles (F1-F5). Androstenedione and estradiol output was measured in incubation media from 100,000 theca cells and progesterone content was determined in the incubation media of 100,000 granulosa cells. All incubations were conducted in 1 ml of Medium 199 at 37 degrees. Steroid output was quantitated by radioimmunoassay of incubation media. Potency estimates were derived by calculation of a peak stimulation index. The standard reference preparation was bLH (NIAMDD-LH-B4). Steroidogenesis was stimulated by three avian LH preparations. preparations. PRC AE1-1 was the most potent, with IRC-2 and B150A showing approximately 50% of the biological activity of PRC AE1-1 in most tissues. Turkey LH HS-5-18 was generally not potent. The presence of multiple isohormones of LH was implied, as various LH preparations exhibited different potency estimates in different tissues. The effects of FSH on steroidogenesis were not significant in most cases. Although the addition of cFSH AGCQSQ113445C failed to significantly increase output of estradiol from small follicles, potency estimates of this preparation were 0.15, 0.20, and 0.13 relative to NIAMDD-LH-B4 follicles was more highly stimulated by LH than by FSH, and thus it would seem that FSH does not play a significant role in steroidogenesis in the hen's ovary. The results of this study suggest that steroid biosynthesis in the hen's ovary may be regulated by multiple forms of LH.