Neonatal maternal separation alters learning and memory. Glucocorticoids also modulate adult learning and memory, and neonatal maternal separation alters forebrain glucocorticoid receptor (GR) concentrations. We used eyeblink classical conditioning to assess the effect of neonatal maternal separation on associative learning. We assessed delay eyeblink conditioning, GR expression, and total neuron number in the interpositus nucleus, a critical site of plasticity in eyeblink conditioning, in adult rats that had undergone either standard animal facilities rearing, handling for 15 min, or maternal separation for either 15 or 60 min per day on postnatal days 2-14. At 2-3 months of age, delay eyeblink classical conditioning was assessed. Brains were processed for GR immunohistochemistry, and GR expression in the interpositus nucleus was assessed using a computer-based densitometry system. Neuron counts and nuclear volumes were obtained from an alternate series of thionin-stained sections. Maternal separation significantly impaired eyeblink conditioning in male but not female rats. Handling and maternal separation did not significantly affect interpositus neuron number and volume. However, prolonged maternal separation significantly increased GR expression in the posterior interpositus in males, and increases were correlated with eyeblink conditioning. In female rats, maternal separation and handling did not significantly alter interpositus neuron number, volume, or GR protein expression, and GR expression did not correlate with eyeblink conditioning. Thus, neonatal maternal separation produces adult deficits in eyeblink conditioning and alterations in GR expression in its neural substrate in a sex-dependent manner.