A number of different neuroactive substances have been found in striatal projection neurons and in fibers and terminals in their target areas, including substance P (SP), enkephalin (ENK), and dynorphin (DYN). In a preliminary report on birds and reptiles, we have suggested that SP and DYN are to a large extent found in the same striatal projection neurons and that ENK is found in a separate population of striatal projection neurons. In the present study, we have examined this issue in more detail in pigeons and turtles. Further, we have also explored this issue in rats to determine whether this is a phylogenetically conserved feature of basal ganglia organization. Simultaneous immunofluorescence double-labeling procedures were employed to explore the colocalization of SP and DYN, SP and ENK, and ENK and DYN in striatal neurons and in striatal, nigral, and pallidal fibers in pigeons, turtles, and rats. To guard against possible cross-reactivity of DYN and ENK antisera with each others' antigens, separate double-label studies were carried out with several different antisera that were specific for DYN peptides (e.g., dynorphin A 1-17, dynorphin B, leumorphin) or ENK peptides (leucine-enkephalin, metenkephalin-arg6-gly7-leu8, methionine-enkephalin-arg6-phe7). The results showed that SP and DYN co-occur extensively in specific populations of striatal projection neurons, whereas ENK typically is present in different populations of striatal projection neurons. In pigeons, 95-99% of all striatal neurons containing DYN were found to contain SP and vice versa. In contrast, only 1-3% of the SP+ striatal neurons and no DYN neurons contained ENK. Similarly, in turtles, greater than 75% of the SP+ neurons were DYN+ and vice versa, whereas ENK was observed in fewer than 5% of the SP+ neurons and 2% of the DYN+ neurons. Finally, in rats, more than 70% of the SP+ neurons contained DYN and vice versa, but ENK was found in only 5% of the SP+ neurons and in none of the DYN+ perikarya. Fiber double-labeling in the striatum and its target areas (the pallidum and substantia nigra) was also consonant with these observations in pigeons, turtles, and rats. These results, in conjunction with studies in cats by M.-J. Besson, A.M. Graybiel, and B. Quinn (1986; Soc Neurosci. Abs. 12:876) strongly indicate that the co-occurrence of SP and DYN in large numbers of striatonigral and striatopallidal projection neurons in a phylogenetically widespread, and therefore evolutionarily conserved, feature of basal ganglia organization.(ABSTRACT TRUNCATED AT 400 WORDS)