Oral etonitazene self-administration was systematically investigated in two inbred strains of rats, Lewis (LEW) and Fischer 344 (F344). For LEW rats, etonitazene maintained higher rates of lever pressing and was consumed in larger volumes than the water vehicle when the reinforcement schedule was fixed ratio (FR) 8. In contrast, with F344 rats responding did not systematically exceed water values at any etonitazene concentration. LEW rats also drank substantially more etonitazene than F344 rats, and at FR 8 only LEW rats showed the typical inverted U-shaped function between etonitazene concentration and number of responses. For the LEW strain, response rate increased as FR size increased from FR 1 to FR 2 and FR 4, but decreased at FR 8. For the F344 strain, as FR size increased response rate showed small increases, but the response rates were far lower than those of the LEW strain. The results support the conclusion that etonitazene was an effective reinforcer for LEW but not F344 rats. These findings demonstrate genetic differences in opioid reinforcement of operant behavior and indicate that genotype can be an important determinant of whether etonitazene serves as a reinforcer.