The present study examined the pharmacology of dihydromorphine, 6-acetyldihydromorphine and dihydroheroin (3,6-diacetyldihydromorphine). Like morphine, dihydromorphine and its acetylated derivatives all were highly selective mu-opioids in receptor binding assays. All the compounds were potent mu-selective analgesics, as shown by their sensitivity towards the mu-selective opioid receptor antagonists naloxonazine and beta-funaltrexamine. However, the actions of dihydromorphine and its analogs were readily distinguished from those of morphine, differences that were surprising in view of the very limited structural differences among them that consisted of only the reduction of the 7,8-double bond. Like heroin and morphine-6beta-glucuronide, the analgesic actions of dihydromorphine and its two acetylated derivatives were antagonized by 3-O-methylnaltrexone at a dose that was inactive against morphine analgesia. Antisense mapping also distinguished between morphine and the dihydromorphine compounds. Antisense oligodeoxynucleotides targeting exon 2 of the cloned MOR-1 gene decreased dihydromorphine analgesia and that of its acetylated derivatives, but not morphine analgesia. Conversely, the exon 1 antisense that effectively lowered morphine analgesia was inactive against dihydromorphine and its analogs. Finally, dihydromorphine and its analogs retained their analgesic activity in a mouse model of morphine tolerance, consistent with incomplete cross-tolerance. Together, these findings imply that the mu-opioid receptor mechanisms mediating the analgesic actions of dihydromorphine and its acetylated analogs are distinct from morphine and more similar to those of heroin and morphine-6beta-glucuronide.