GABAergic and enkephalinergic afferents to the ventral tegmental area were investigated in the rat using retrograde tracing techniques combined with in situ hybridization. Following iontophoretic deposit of Fluoro-Gold in the ventral tegmental area labeling in the forebrain was most dense in the shell of the nucleus accumbens, rostral ventromedial ventral pallidum and diagonal band of Broca. A smaller density was also observed in the lateral septum. In these forebrain regions, the portion of retrogradely labeled cells that contained mRNA for glutamate decarboxylase ranged from 25% to 50%, whereas only 5% to 15% were double-labeled for preproenkephalin mRNA. Cells double-labeled with either glutamate decarboxylase or preproenkephalin mRNA were most numerous in the lateral septum, shell of the nucleus accumbens, rostral ventral pallidum and diagonal band of Broca. Large Fluoro-Gold deposits which invaded the medial substantia nigra resulted in a significant number of retrogradely labeled cells in the core of the nucleus accumbens, and a portion of these neurons also contained mRNA for glutamate decarboxylase or preproenkephalin. These data demonstrate the presence of GABAergic and enkephalinergic neurons projecting from the nucleus accumbens, ventral pallidum and diagonal band of Broca to the ventral tegmental area.