It is known that testicular interstitial fluid (TF) contains thermolabile factors that can alter in vitro production of androgens by the Leydig cells. The net stimulatory activity of this fluid increases in association with the disruption of spermatogenesis. The identity of the active agent(s) in TF is not known. Therefore, the authors used gel-liquid chromatography to initially characterize TF from control and bilaterally cryptorchid animals. The stimulatory activity of TF was retained on Concanavalin A Sepharose columns. Gel filtration on Ultrogel AcA 44 suggested a molecular size between 40 and 90 kD. The unfractionated fluid from control and bilaterally cryptorchid rats, as well as the chromatographic fractions containing stimulatory activity, were further resolved by SDS-PAG electrophoresis. At least three bands representing glycoproteins with apparent molecular size between 57 and 75 kD were seen in all samples containing stimulatory activity. No difference in the pattern of protein bands was seen between TF from control and bilaterally cryptorchid testes. However, samples reduced with beta-mercaptoethanol showed protein bands with apparent molecular size of 78 and 118 kD which were present only in unpurified control TF. These data support the possibility that the stimulatory substance in TF from control and bilaterally cryptorchid testes is a glycoprotein with a molecular size between 57 and 75 kD. Differences in the bioactivity of the unfractionated TF may be due in part to the presence of additional larger protein molecules in the control TF.