Bioassay and immunological studies have detected the presence of opioid peptides in the nervous system of representatives of all classes of vertebrates. The present study evaluates the expression and localization of preproenkephalin (PPE) mRNA to determine the sites of synthesis of the enkephalin peptides in the adult chicken and pigeon telencephalon using in situ hybridization histochemistry. We used a 500-base-pair chicken RNA probe corresponding to chicken PPE cDNA. In both the chicken and the pigeon telencephalon, the highest concentration of PPE mRNA-containing cells was observed in the lobus parolfactorius, paleostriatum augmentatum, nucleus accumbens, and septum. Distinct populations of labeled cells were also detected in the hyperstriatum accessorium, hippocampus, area parahippocampalis, nucleus of the diagonal band, cortex dorsolateralis, and cortex piriformis. Differences in PPE mRNA expression between chicken and pigeon were observed in several telencephalic regions. For instance, the bulbus olfactorius was heavily labeled in the pigeon, but was not labeled in the chicken, and numerous PPE mRNA-containing cells were present in the area parahippocampalis of pigeons but not of chickens. In contrast, in the hyperstriatum dorsale and hyperstriatum ventrale, numerous PPE mRNA-expressing cells were detected in the chicken but not in the pigeon. Overall, PPE mRNA-expressing cells were more numerous than enkephalin-immunoreactive cells described in previous studies. In addition, our results suggest that the general pattern of enkephalin expression in the avian telencephalon is similar to that found in other vertebrates. Finally, the results of the present study illustrate some differences in the pattern of PPE mRNA distribution between closely related species, indicating the existence of species-specific neurochemical pathways, which may influence and perhaps mediate different behaviors characteristics of these species.