The mu opioid receptor may constitute a critical component of a negative feedback system that regulates Pavlovian fear conditioning. We investigated context fear conditioning acquisition and expression in mu opioid receptor knockout mice (on an inbred, C57 genetic background). We discovered that the mu receptor knockout results in an unexpected and significant deficit in context fear acquisition. Mice lacking the mu receptor showed normal fear acquisition when subjected to a 1-day fear conditioning protocol but evinced deficient fear learning when acquisition was conducted across 5 days. The knockout mice showed normal reactivity to footshock in both fear conditioning protocols. Finally, we confirmed the effectiveness of the receptor deletion in the C57 strain by subjecting the mice to a test of morphine analgesia in the hot-plate assay. As has been seen with mixed genetic background, the receptor deletion resulted in a complete lack of analgesic response to 10 mg/kg morphine. Surprisingly, mice with a single copy of the mu receptor gene (heterozygous knockouts) showed intact sensitivity to morphine but a significant deficit in Pavlovian fear conditioning. The results indicate that deletion of the mu receptor gene impairs fear conditioning and that the conditioning and analgesia effects of heterozygous deletion are dissociable. The conditioning deficit seen in this line of mice may be related to impairment in hippocampus function.