Rats were exposed to short-term restraint (held by the tail for 1 min), injected s.c. with saline or subjected to the combination of these treatments. Fifteen and 30 min after these treatments the means serum corticosterone level was significantly increased by more than four times, compared to rats taken directly from their home cages, indicating a stress response. In the peri-aqueductal grey, the level of substance P-like immunoreactivity was increased by 45% (P < 0.01) and 65% (P < 0.01) 30 and 60 min after the combined treatment, respectively. Significant increases of the level of substance P-like immunoreactivity in the peri-aqueductal grey were also found after restraint only and after a s.c. saline injection. Similar, but less marked, changes in the level of cholecystokinin-like immunoreactivity in the PAG were also seen. In the accumbens a significantly decreased level of substance P-like immunoreactivity was encountered at 15 and 30 min after treatment, while the levels of cholecystokinin- and neuropeptide Y-like immunoreactivity were not significantly changed. In other regions studied, no effects on peptide levels were seen. The changes in peptide levels had a time course similar to that of the increase in serum corticosterone. Also the successive removal of rats from a common cage was found to increase significantly the serum corticosterone and the substance P-like immunoreactivity in the peri-aqueductal grey in the animals that were taken late in sequence from the cage.(ABSTRACT TRUNCATED AT 250 WORDS)