The inhibitory feedback effects of steroids on pituitary LH release are believed to be mediated via steroidal effects on the hypothalamic LHRH activity. We have examined the direct effects of individual steroids (T, DHT and E2) on hypothalamic LHRH levels and on LHRH release in vitro. In castrated male rats, replacement of either steroid in physiological doses, resulted in augmentation of the MBH LHRH levels by steroidal action within the MBH. LHRH analyses of microdissected diencephalic nuclei revealed that this accumulation occurred exclusively in LHRH terminals in the ME. Careful examination of the time course of steroid action showed that whereas LH release was suppressed within hours of steroid treatment, the LHRH response occurred after 3-4 days of steroid exposure in 2-week castrated rats and 7-14 days in 8-week castrated rats. This temporal dichotomy in the LH and LHRH responses to steroid action was further substantiated by the differential effects of low, sub-physiological levels of steroids on these two responses. Very low levels of T or E2 evoked maximal accumulation of the MBH LHRH, but LH release in vivo and the rate of LHRH release in vitro were not affected. Surprisingly, physiological levels of T which suppressed LH release concomitant with elevations in LHRH levels, augmented the in vitro rate of LHRH release. In fact, the LHRH release rate was found to be correlated with LHRH concentrations in hypothalami of intact, castrated and castrated rats treated with T. Thus it appears that in the hypothalamo-pituitary axis there are different thresholds of responsiveness to steroids. Apparently, the LHRH neurons, particularly the processes involved in LHRH accumulation are most sensitive to low levels of steroids; however, higher physiological levels of steroids are required to suppress pituitary LH release as well as to promote LHRH release. On the basis of our cumulative data, it is reasonable to speculate that steroid-induced accumulation of LHRH in the ME may not be a consequence of decrease in LHRH release, but may involve synthesis of the neurohormone.