The use of testicular interstitial fluid (IF) collected from the rat testis has been validated as (1) an index of the total extracellular extratubular fluid volume of the testis (which reflects the permeability of testicular capillaries) and (2) as a means of measuring changes in the interstitial hormonal environment. The former was tested by comparing the albumin 'space' with the volume of recovered IF in the same testes from control and bilaterally cryptorchid rats sampled at 0-40 h after injection of hCG. Although the volume of IF recovered was on average only 50% of the albumin 'space', both measures increased in parallel after hCG injection and were always closely correlated (P less than 0.001) over a 4- to 5-fold range. The volume of recovered IF increased with age in parallel with increase in testicular weight, and the testosterone concentration in IF paralleled changes in peripheral serum, increasing from 45 to 80 days of age and then declining. After injection of 25 micrograms bovine LH, testosterone levels in IF, spermatic venous (SV) and peripheral venous (PV) blood increased up to 10-fold by 1 h and returned to control levels over the next 11 h. Testosterone levels in IF were always considerably higher than those in SV blood, but this difference was not constant. Subcutaneous injection of rats with an LH-RH agonist resulted in parallel increases in the serum levels of LH and in the IF and PV levels of testosterone. However, at 6 h there was an 'LH-independent' secondary increase in testosterone levels which was associated with an increase in IF volume, reflecting an increase in capillary wall permeability and hence increased transport of LH into the testis.