The domestic hen with its well-organized follicular hierarchy offers a unique model system for the study of the role of inhibin in follicular development. We have validated a homologous bovine RIA for use in the hen to measure plasma immunoreactive inhibin-like activity during the ovulatory cycle as well as during experimental conditions in which follicular development has been altered. Validation of the bovine RIA kit revealed that increasing amounts of hen plasma gave linear increases in the amount of immunoreactive inhibin detected. This assay system as well as a homologous chicken LH RIA were used to assess plasma concentrations of immunoreactive inhibin and LH, respectively, in experiments 1-3. In experiment 1, blood samples were collected from hens (n = 5) at 2-h intervals for 24 h starting at 1600 h after the lay of the last egg of the sequence through the following day. No significant peak in plasma immunoreactive inhibin level was detected during the ovulatory cycle, in spite of a significant preovulatory rise in plasma LH. The highest level of plasma immunoreactive inhibin was attained, however, 2 h prior to the preovulatory LH surge. In experiment 2, eCG (n = 6) or vehicle (n = 3) was administered to hens daily for 6 days. A blood sample was drawn immediately before injection each day as well as 24 h after the last day of treatment. Treatment with eCG significantly (p < 0.01) raised plasma immunoreactive inhibin-like levels, compared to vehicle-injected controls. In experiment 3, three to four of the largest follicles (F1-F4) were surgically removed, or sham surgery was performed.(ABSTRACT TRUNCATED AT 250 WORDS)