Intratesticular tissue fluid (ITF) was obtained by centrifugation of the decapsulated testis of the adult rat. Testosterone level in ITF was increased by direct injection of LH [luteinizing hormone] into the testis and was lowered by anti-LH serum. Based on the result of previous reports with testosterone in testicular vein blood, a simplification of the LH bioassay without impairing sensitivity and accuracy was undertaken using ITF as the material for testosterone measurement. Dose-response relationship between injected LH and testosterone concentration in ITF was observed. The sensitivity of the assay was 5 ng for LH (NIH-LH-S15). FSH [follicle-stimulating hormone] (NIH-FSH-S10) and prolactin (NIH-P-S11) had no detectable activity on testosterone production. This assay method depending on biological activities of LH will be helped for unbiased evaluation of the result obtained by radioimmunoassay.