As intratesticular communication between the Sertoli and Leydig cells must take place via testicular interstitial fluid (IF), we have tested whether this fluid contains a factor(s) which has effect on testosterone secretion by Percoll-purified Leydig cells in vitro. Addition of increasing concentrations of charcoal-stripped IF to Leydig cells caused dose-dependent stimulation of basal testosterone secretion and greatly enhanced the response to a maximally stimulating dose of hCG. These effects were first evident after 2 h of incubation and were progressive up to 24 h. Charcoal-stripped serum from the same donor animals also had some stimulatory effect on basal and, occasionally, on hCG-stimulated testosterone secretion, but the magnitude and pattern of this effect and its limited dose-dependence were very different from the effects seen with IF. The effects of IF and serum were not altered by the addition of an antiserum to LH. It is therefore concluded that testicular IF contains a factor(s), not derived from serum, which can alter Leydig cell testosterone responsiveness. This factor(s) was heat-sensitive with a MW of greater than 10 000 daltons. IF from the abdominal testes of adult rats which had been made unilaterally cryptorchid contained much higher levels of the stimulatory factor(s) compared to the contralateral scrotal testes, and this is of interest because of the well-documented alteration in Sertoli-Leydig cell interaction that occurs in this situation. These results provide the first direct evidence that one or more factors capable of altering testosterone secretion are produced within the testis of the normal adult rat, and raises the possibility that this factors(s) may be involved in the local regulation of the intratesticular testosterone levels.