Measurements of immunoactive and bioactive serum LH in the rat were performed after optimization of both RIA and rat interstitial cell-testosterone (RICT) assay to measure the low circulating LH levels of intact male rats. The effective sensitivity of the RIA was increased 3- to 5-fold to 6.2 or 4.7 ng RP1/ml by extraction of 1--1.5 ml serum, respectively. Serum LH levels of adult male rats were 48.5 +/- 16.3 ng RP-1/ml (n = 50), became undetectable after hypophysectomy, and were suppressed 50% after estrogen treatment. For RICT assay of serum rat LH, sensitivity was increased 5-fold by reduction of the incubation volume to 0.35 ml containing 2--4 x 10(6) interstitial cells/tube, with a detection limit of 3 ng RP-1 or 0.1 ng pure LH. The within-assay coefficient of variation for measurement of a pool of control male rat serum (126 +/- 19l6 ng RP-1) WAS +/- 12.5% and the between-assay variation was +/- 15%. Bioactive serum LH levels in adult male rats were 124.8 +/- 32.3 ng RP-1/ml or 2.74 +/- 0.71 ng rat LH/ml, with biopotency to immunopotency (B:I) ratios of 2.8 +/- 0.2 and 1.1 +/- 0.1, respectively, that were unchanged during elevations of serum LH by LHRH administration. Pituitary LH content was 726 +/- 183 micrograms RP-1 or 16.4 micrograms rat LH/gland, with B:I ratios of 2.3 +/- 0.4 and 1.0 +/- 0.2, respectively. The difference between B:I ratios in assays employing pure rat LH and assays using the RP-1 preparation was attributable to the presence of alpha-subunit and biologically inactive material in the RP-1 standard. Precise measurements of immunoactive and bioactive rat LH in male rat plasma can not be performed by these modified RICT and RIA procedures. The sensitive RICT assay can also be applied to the measurement of low levels of LH in the serum of primates and other species.