In the liver, function of the bile salt export pump (Bsep), a major canalicular exporter of mammalian liver, is complemented by activity of the multidrug resistance protein 2 (Mrp2), a canalicular organic anions transporter. The latter membrane protein was found capable of transporting a wide range of anticancer drugs out of cells, eventually undermining their therapeutic potential and contributing to multidrug resistance. Because understanding of the function of Bsep and Mrp2 may have important clinical implications, we employed a reverse transcription polymerase chain reaction, immunoblotting, and immunofluorescence to examine their gene, protein expression, and distribution of antigenic sites in the rat liver during development from 16-day-old fetus to adult animal. Bsep mRNA was almost undetectable before birth. It was first clearly expressed in the liver of newborn rats. In postnatal period, expression of Bsep mRNA showed a steady increase. On the contrary, Mrp2 mRNA was seen before birth, altough at low levels. In concert with mRNA expression, Bsep protein was undetectable before birth, while Mrp2 protein was already expressed. Both proteins were clearly detectable in postnatal period. Confocal immunofluorescent microscopy showed asynchronous appearence of Bsep and Mrp2 during development, but their colocalization in the bile canaliculi once each one is expressed. During gestational period, a weak immunofluorescence for Mrp2 was observed only in livers of 16-day-old embryos. Anti-Mrp2 antibody stained the nascent bile canaliculi. No fluorescence for Bsep was seen. Both proteins were clearly visualizable after birth, although the pattern of immunostaining varied. These findings provide molecular evidence that expression of both Bsep and Mrp2 during development is transcriptionally regulated. They also point out to the different relevance to the liver function of the systems responsible for canalicular transport of bile salts versus organic anions.