The effect of exposure to nitrous oxide (N2O) on the levels of preproenkephalin mRNA in the hypothalamus of rats was examined. In the first experiment, rats were exposed to 1000 ppm N2O for 8 h a day over 4 days. Compared with controls (which were exposed to air over the same duration), the N2O exposed animals exhibited significant elevations in preproenkephalin mRNA levels in the hypothalamus. In a second experiment, rats were exposed to 60% N2O or air for 12, 24 and 48 h duration, and hypothalamic levels of preproenkephalin mRNA as well as methionine enkephalin were analyzed. Compared with controls, N2O exposed rats exhibited significant elevations in preproenkephalin mRNA levels. The levels on preproenkephalin mRNA were significantly higher after 48 h of N2O exposure than after 12 h of N2O exposure. Similarly, the concentration of methionine enkephalin was significantly higher after 24 and 48 h of exposure of N2O than after exposure to 12 h of N2O or air. These results indicate that (a) exposure to N2O results in significant elevations in preproenkephalin mRNA levels, (b) the increased preproenkephalin mRNA levels appear to be proportional to the concentration of N2O exposure as well as the duration of N2O exposure, and (c) N2O-induced elevation in preproenkephalin mRNA levels is associated with corresponding increase in tissue concentrations of methionine enkephalin. In total, these results suggest that N2O selectively stimulates synthesis of methionine enkephalin in the diencephalic region of the brain.