Abstract Acute cocaine administration increases phosphorylation of dopamine and cAMP-regulated phosphoprotein (M(r) 32 kDa) (DARPP-32) at threonine (Thr)-34, whereas repeated cocaine administration increases DARPP-32 phosphorylation at Thr-75 in Sprague-Dawley rat striatum. Repeated acetyl-l-carnitine (ALCAR) administration persistently increases dopamine outflow in the nucleus accumbens. The present study examined the effect of repeated ALCAR administration on the DARPP-32 phosphorylation pattern in the nucleus accumbens and caudate-putamen. ALCAR increased phosphoThr-34 DARPP-32 levels and decreased phosphoThr-75 DARPP-32 levels, after 1 and 10 days of washout. We compared the effects of repeated cocaine and repeated ALCAR administrations on the behavioural response to cocaine challenge and on the DARPP-32 phosphorylation pattern and cyclin-dependent kinase 5 (Cdk5) levels in the striatum. We also studied whether ALCAR administered daily during or after cocaine sensitization procedure would interfere with the effects of cocaine. When the response to the cocaine challenge was assessed, cocaine- and ALCAR-treated rats showed a similar sensitized behavioural response, and rats receiving combined cocaine and ALCAR treatments, irrespective of treatment order, also showed a sensitized response. A week after the cocaine challenge, the two drugs had induced opposite modifications in DARPP-32 phosphorylation, as cocaine increased phosphorylation at Thr-75, while ALCAR increased phosphorylation at Thr-34. In cocaine plus ALCAR treated rats, irrespective of treatment order, ALCAR administration antagonized cocaine effects on DARPP-32 phosphorylation. Moreover, cocaine, but not ALCAR, increased DeltaFosB and Cdk5 expression, and the increase in Cdk5 was antagonized by ALCAR administration in rats receiving combined treatments. These effects were relatively persistent, as they were still present 7 days after the last treatment.