Variations in the concentration of free calcium in neurons is believed to play a major role in regulating neuronal excitability. Because calcium-binding proteins such as calbindin D-28k and calretinin help to regulate intracellular calcium, we investigated the possibility that the expression of these proteins may be affected in genetically epilepsy-prone rats (GEPRs). The mRNA levels of both proteins were compared across several brain regions using in situ hybridization histochemistry and Northern blot analysis with semiquantitation by optical density measures in autoradiograms from two GEPR strains that differ in the severity of audiogenic seizures (GEPR9 and GEPR3) and from Sprague-Dawley rats. Results revealed a lower level of expression in calbindin D-28k mRNA in the in the caudate putamen-accumbens nuclei in GEPR3 (-30%) and GEPR9 (-60%) relative to controls. The calbindin D-28k mRNA level was also lower in the reuniens nucleus of the thalamus (-41% in GEPR3; -34% in GEPR9). The calretinin mRNA level was lower in the substantia nigra compacta of both GEPR rat strains (-31% in GEPR3 and -34% in GEPR9 relative to controls). No changes in mRNA were detected in other brain regions expressing calbindin D-28k or calretinin mRNA. These results indicate that the expression of these related calcium-binding proteins is altered in the GEPRs before the induction of seizures. This initial defect could alter either the calcium-buffering capacity or regulation of calcium-mediated processes by these proteins and thus play a role in the molecular cascade of events inducing the genetic susceptibility to, and the generalization of, seizures in these rat strains.