In the present study, we investigated the effects of lithium on endogenous ADP-ribosylation in rat brain. It was found that addition of lithium in vitro inhibits endogenous ADP-ribosylation activity in extracts of frontal cortex at therapeutically relevant concentrations. Inhibition is observed at concentrations as low as 0.3 mM and is maximal at 1 mM when 50% inhibition is obtained. A similar degree of inhibition of endogenous ADP-ribosylation was observed for all substrate proteins identified, including Gs alpha, suggesting that lithium's effect may be achieved at the level of ADP-ribosyltransferases and not specific substrate proteins. In contrast to lithium, chloride salts of sodium and potassium do not alter endogenous ADP-ribosylation activity in frontal cortex. To assess the possible in vivo relevance of this in vitro action of lithium, we studied the effect of chronic lithium administration on levels of endogenous ADP-ribosylation in frontal cortex. It was found that chronic lithium treatment, in contrast to the inhibitory effect of the drug in vitro, produced a > 35% increase in endogenous ADP-ribosylation activity. A similar degree of increase was observed for all of the substrate proteins identified. These novel findings raise the possibility that certain endogenous ADP-ribosyltransferases are among the acute targets of lithium in the brain and that adaptations in these enzymes may be part of the mechanisms underlying lithium's long-term effects on brain function.