The purpose of this study was to compare the heterogeneity and median charge of FSH and LH in serum at different phases of the normal human menstrual cycle. Serum specimens were obtained during the follicular phase [9.0 +/- 1.9 (+/- SD) days before the midcycle], at the midcycle LH peak, and during the luteal phase (9.75 +/- 1.6 days after the midcycle) in 16 women with normal menstrual cycles. The 48 serum specimens were subjected to electrophoresis in 0.17% agarose suspension in 0.075 M veronal buffer at pH 8.6, using a column suitable for measuring the median charge of the isoforms of FSH and LH, expressed as median electrophoretic mobility. Four sera were also analyzed by use of a larger column, giving a high resolution. The FSH and LH activities were measured with a time-resolved sandwich fluoroimmunoassay. The number of isoforms of both FSH and LH in each serum specimen analyzed by electrophoresis with high resolution was between 20-30. The median charge of the isoforms of FSH was less negative (P < 0.001) at the midcycle than in the follicular or luteal phase in all 16 women. The same was found for LH in 14 of 15 women. The median charge of FSH or LH in the follicular phase was not significantly different from that in the luteal phase of the cycle. We conclude that at least 20-30 isoforms of both FSH and LH circulate in blood during the menstrual cycle. More basic isoforms of both hormones appear in serum at midcycle than in the follicular or luteal phase. The difference is most likely due to a selective secretion from the pituitary of more basic forms of FSH and LH at midcycle.