At least two drug efflux pumps involved in multidrug resistance, P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (Mrp1), are expressed in rat astrocyte primary cultures. The aim of this study was to compare the expression of P-gp and Mrp1 in primary cultures exposed to 50 or 500 ng/mL doxorubicin (DOX). Among the two P-gp genes expressed in rodents, mdr1a and mdr1b, a time- and dose-dependent increase in mdr1b mRNA levels was revealed by northern blot analysis. This up-regulation was inhibited by actinomycin D and occurred as early as 2 h after exposure to 50 or 500 ng/mL DOX, whereas mdr1a and mrp1 transcripts were not modified by the DOX exposure. In addition, DOX also strongly enhanced, in a time- and dose-dependent manner, P-gp but not Mrp1 expression. Moreover, DOX raised the cellular efflux of vincristine, a substrate for both P-gp and Mrp1. This efflux was inhibited by the P-gp modulators PSC833 and GW918, but not by the Mrp1 modulator MK571. On the other hand, a 24-h exposure to 500 ng/mL DOX, but not 50 ng/mL DOX, induced apoptosis in primary cultures of rat astrocytes. Fumonisin B1, a ceramide synthase inhibitor, reduced DOX-induced apoptosis, suggesting that de novo synthesis of the ceramide regulatory pathway might be involved in DOX-induced apoptosis. Moreover, western blot analysis showed that fumonisin B1 was not able to decrease the overexpression of P-gp induced by DOX. Our results provide evidence that DOX up-regulates a functional P-gp in primary cultures of rat astrocytes and might cause astrocyte apoptosis via the ceramide pathway.