The hippocampal formation (HF) is involved in modulating learning related to drug abuse. While HF-dependent learning is regulated by both endogenous opioids and estrogen, the interaction between these two systems is not well understood. The mossy fiber (MF) pathway formed by dentate gyrus (DG) granule cell axons is involved in some aspects of learning and contains abundant amounts of the endogenous opioid peptide dynorphin (DYN). To examine the influence of ovarian steroids on DYN expression, we used quantitative light microscopic immunocytochemistry to measure DYN levels in normal cycling rats as well as in two established models of hormone-treated ovariectomized (OVX) rats. Rats in estrus had increased levels of DYN-immunoreactivity (ir) in the DG and certain CA3 lamina compared with rats in proestrus or diestrus. OVX rats exposed to estradiol for 24 h showed increased DYN-ir in the DG and CA3, while those with 72 h estradiol exposure showed increases only in the DG. Six hours of estradiol exposure produced no change in DYN-ir. OVX rats chronically implanted with medroxyprogesterone also showed increased DYN-ir in the DG and CA3. Next, dual-labeling electron microscopy (EM) was used to evaluate the subcellular relationships of estrogen receptor (ER) alpha-, ERbeta and progestin receptor (PR) with DYN-labeled MFs. ERbeta-ir was in some DYN-labeled MF terminals and smaller terminals, and had a subcellular association with the plasmalemma and small synaptic vesicles. In contrast, ERalpha-ir was not in DYN-labeled terminals, although some DYN-labeled small terminals synapsed on ERalpha-labeled dendritic spines. PR labeling was mostly in CA3 axons, some of which were continuous with DYN-labeled terminals. These studies indicate that ovarian hormones can modulate DYN in the MF pathway in a time-dependent manner, and suggest that hormonal effects on the DYN-containing MF pathway may be directly mediated by ERbeta and/or PR activation.