Calbindin-D(28K) (CALB), a calcium-binding protein, is thought to buffer intracellular calcium levels in neurons playing several important roles during central nervous system development such as, protecting against apoptosis (programmed cell death), neurodegenerative diseases and influencing sexually dimorphic brain structures. While preliminary research indicates that calbindin levels are modulated by steroids, there has not yet been a comprehensive study to determine the role androgens, androgen metabolites and corticosterone have on CALB expression in males and throughout the estrous cycle in rats. The present study had two main components utilizing Western analysis: (1) examination of the influence of steroid hormones (testosterone, dihydrotestosterone, corticosterone, and estradiol) and aromatase and 5alpha-reductase blockers on rat brain CALB expression in frontal cortex, MBH-POA, and cerebellum of male rats in experiment 1; (2) characterization of CALB abundance in frontal cortex, MBH-POA, and cerebellum throughout the hormonally induced estrous cycle of rats in experiment 2. There were no significant alterations in CALB levels by any of the treatments in experiment 1 or experiment 2 when frontal cortical tissue was examined. In male MBH-POA samples, estradiol treatment significantly increased CALB levels compared to oil-injection and aromatase or 5alpha-reductase blockers. Additionally, DHT treatment significantly increased CALB levels vs. 5alpha-reductase blocker values. In female samples, MBH-POA CALB levels increased from diestrus through proestrus and estrus to metestrus, where metestrus values were significantly higher compared to diestrus levels. In the cerebellum, the only significant alteration in CALB levels, in males, was observed in corticosterone-treated animals where a significant decrease was seen compared to oil injection. In females, cerebellum CALB levels increased from diestrus through proestus, with a slight decrease at estrus. Thereafter, CALB levels increased during metestrus where cerebellar CALB values were significantly higher than diestrus levels. These findings suggest that steroid hormones play an important regulatory role in CALB expression in the brain (except for the frontal cortex) where it potentially influences the development/function and neuroprotective changes of neuroanatomical structures.