The pituitary gonadotropin response of synthesis and release to two step stimulation at a 60 minute interval with 100 micrograms of luteinizing hormone-releasing hormone (LH-RH) was studied by radioimmunoassay of hLH and hFSH in the normal menstrual cycle, amenorrhea and anovulation. The definition of delta 1 and delta 2 was as follows: delta 1 = 30 (or 60) min. value-0 min. value. delta 2 = 90 (or 120, 180) min. value-60 min. value. delta 1, delta 2 ratio = delta 2/delta 1. Serum mean levels of hLH in follicular and luteal phase increased after the first injection of LH-RH. The second injection of the hormone induced an increase to above the first peak, indicating the priming effect of the first LH-RH on the second peak. The first peak is presumed to be the pituitary gonadotropin release due to the first injection of LH-RH, and the second peak can be composed in part of hLH newly synthesized by the first LH-RH stimulation. The response of hLH in the ovulatory phase increased after the first administration, but showed no significant peak after the second. The LH-RH two step test in normoestrogenic and PCO type amenorrhea produced two peaks of the mean levels of hLH. In ovarian anovulation, an hLH value higher than the others was detected before the first LH-RH stimulation. The first injection caused an increase in serum hLH but after the second there is no significant peak. In contrast, serum hLH and hFSH values were lower than in the normal menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)