GnRH neuronal activity is increased during the proestrous LH surge as assessed by the expression of cFos. Whether there is an increase in GnRH mRNA concomitant with GnRH neuronal activation is still controversial. In the present study we have compared GnRH mRNA levels in GnRH neurons expressing cFos (activated) with those not expressing cFos during the proestrous LH surge. Brain tissue was obtained from adult female rats perfused on proestrus before (1100-1300 hr) or during (1500-1900 hr) the LH surge. Twenty-five microns free-floating brain sections were subjected to double labeling of cFos by immunocytochemistry and of GnRH mRNA by in situ hybridization using an 35S-labeled cRNA probe. Another series of sections were stained by double-label immunocytochemistry for cFos and GnRH. Serum LH concentrations and cFos expression in GnRH neurons indicated that rats sacrificed at or before 1300 hr had basal levels of LH secretion, and no cFos activity was detected in GnRH neurons. In animals sacrificed between 1500 and 1900 hr, LH concentrations were at levels indicative of the ascending phase of the LH surge. A significant positive correlation between plasma LH values and the proportion of GnRH mRNA-containing neurons expressing cFos was observed. The average cellular GnRH mRNA content (silver grain numbers per cell) during the LH surge was not different from that before the surge. However, if GnRH neurons during the surge were further divided into cFos-positive and cFos-negative neurons, GnRH mRNA levels in the cFos-positive GnRH neurons were significantly increased by 28% when compared with GnRH neurons without cFos expression. These data suggest that cFos expression in GnRH neurons during the proestrous LH surge identifies transcriptionally more active GnRH neurons.