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The regional distribution of immunoreactive delta sleep-inducing peptide (IR-DSIP) and the effect of 24-h sleep deprivation on the IR-DSIP content was investigated in the rat brain by means of enzyme immunoassay (EIA) of the assayable IR-DSIP in the rat brain coeluted with authentic DSIP in the gel filtration. It is thus suggested that the present EIA method preferentially detects fee DSIP and not bound DSIP. IR-DSIP proved to be more concentrated in the limbic structures, although the contents were generally at a low level. Sleep deprivation and subsequent rebound sleep had no significant effect on the brain contents of 'free-form' DSIP.