Steroidogenesis-stimulating activity (SSA) was examined in testicular intertubular fluid from normal, and short-term and long-term (up to 12 months) experimentally cryptorchid rats, using an in vitro Leydig cell bioassay based on testosterone production over 20 h in the presence of a maximum dose of human chorionic gonadotropin. Total fluid volume increased throughout the period of cryptorchidism, while intertubular testosterone concentrations declined. SSA from cryptorchid rats was significantly greater (2- to 3-fold) than normal at all time-points; however, the major increase in activity occurred within the first 4 weeks after treatment. Similar concentrations of lipoproteins were recovered from both untreated and 4-week cryptorchid fluid by density ultracentrifugation, although the bioactivity of the cryptorchid testis lipoprotein fraction was 8-fold higher than the lipoprotein fraction from untreated testes. Moreover, removal of the lipoproteins led to a loss of SSA in the lipoprotein-deficient fraction of the intertubular fluid. Consequently, the in vitro bioassay conditions were modified by addition of a constant level of serum lipoproteins to all assay wells. Employing the lipoprotein-supplemented bioassay, multiple stimulatory and inhibitory activities were resolved by Sephadex G-100 gel filtration in intertubular fluid from both normal and cryptorchid testes: (i) an inhibitory activity eluting in the void volume (> 150 kDa), which decreased after cryptorchidism; (ii) a stimulatory activity (40-80 kDa), which did not appear to be affected by cryptorchidism; (iii) an inhibitory activity (20-40 kDa) which decreased after cryptorchidism, and (iv) a stimulatory activity (12-20 kDa) which increased after cryptorchidism.(ABSTRACT TRUNCATED AT 250 WORDS)