Hippocampal function is known to be estrous-cycle-dependent but information on estrous-cycle-dependent protein expression is limited. It was therefore the aim to study protein levels of the neuronal network over the estrous cycle in the hippocampus of female rats and in males showing protein chemical neuroanatomy in this area. Female and male OFA Sprague-Dawley rats were used and females were grouped to proestrous, estrous, metestrous and diestrous by using vaginal smears. Hippocampal tissue was taken, proteins extracted, run on two-dimensional gel electrophoresis and proteins were identified by mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Spot volumes were quantified with specific software. A Synapsin-1 expression form was differentially regulated between proestrous and diestrous, a Synapsin IIa expression form was differentially regulated between proestrous and metestrous, the sum of ERC-2 proteins organizing the cytomatrix at the nerve terminals active zone was showing sex-dependent levels in the proestrous phase and Neurofilament triplet L protein was differentially expressed between the estrous phase and males. The findings may represent estrous-cycle-dependent hippocampal synaptic function that has been shown already in terms of electrophysiology and neuroanatomy. Neurofilament changes over the estrous cycle may reflect endoskeleton changes over the estrous cycle. We learn from this study, although increasing complexity of protein knowledge, that the estrous cycle and not only the sex per se has to be taken into account for design of future studies and interpretation of previous work at the protein level.