BACKGROUND: In rodents, third-trimester-equivalent alcohol exposure (TTAE) produces significant deficits in hippocampal-dependent memory processes such as contextual fear conditioning (CFC). The present study sought to characterize changes in both behavior and Fos METHODS: On postnatal day 7, mice received either two injections of saline or 2.5 g/kg EtOH spaced 2 h apart. The mice were left undisturbed until they reached adulthood, at which point they underwent CFC. After context exposure on day 2, mice received a tamoxifen injection. Brain tissue was harvested. Slides were automatically imaged using a Zeiss AxioScanner. Manual counts on a priori regions of interest were conducted. Automated counts were performed on the whole brain using the QUINT 2D stitching pipeline. Last, novel network analyses were applied to identify future regions of interest. RESULTS: TTAE reduced context recall on day 2 of CFC. Fos DISCUSSION: These results suggest that CFC is mediated by the limbic memory system and is disrupted following TTAE. Given the increase in CA1 and CA3 activity, a potential hypothesis is that TTAE causes disruptions to memory encoding following day 1 conditioning. Future studies will aim to determine whether this disruption specifically affects the encoding or retrieval of fear memories.