Lesions of the hippocampus attenuate acquisition of the tone-shock contingency in Trace, but not in Delay fear conditioning. These findings suggest that hippocampal regions are differentially involved in these two forms of fear conditioning. The present study was aimed at testing the hypothesis that hippocampal neurons are differentially activated during acquisition and retrieval of Delay versus Trace fear conditioning. Male C57BL/6J mice were exposed to eight tone-shock pairings (in Trace conditioning the shock came 30 s after the tone), and tested for immobility upon reexposure to contextual stimuli or to one tone presentation. Ten brain regions were analyzed by immunohistochemistry for inducible transcription factors (ITF) c-Fos and Zif268 1.5 h after training, context test or tone test. Acquisition of both Delay and Trace fear conditioning produced significant induction of c-Fos in the majority of brain regions analyzed compared to naive control animals. Importantly, Delay fear conditioning caused a higher increase of c-Fos expression in the CA3 region of the hippocampus compared to Trace-trained animals. After cue reexposure, Zif268 levels in the dentate gyrus of the hippocampus were higher in Trace-conditioned than in Delay-conditioned animals. In addition, reexposure-related c-Fos expression in the anterior cingulate cortex was significantly higher in Delay-conditioned animals than in Trace-conditioned animals. The present study confirms differential activation of hippocampal subregions in Delay and Trace fear conditioning.