l-Carnitine must be transported against a substantial concentration gradient across the epididymal epithelium to achieve high intraluminal levels, approximately 50 mM in the cauda. Recently, an organic cation transporter, OCTN2, was cloned from rat intestinal epithelium and shown to transport l-carnitine in a sodium-dependent manner. To test the hypothesis that OCTN2 was present in the epididymis, primers were designed based on the published OCTN2 mRNA sequence. A 1.9-kilobase OCTN2 cDNA from rat epididymis was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned. Northern analysis demonstrated the presence of OCTN2 transcripts in the epididymis, with highest expression in the distal caput and corpus. To localize the protein, an antibody raised against a carboxy-terminal peptide of OCTN2 was produced in rabbits and used for Western blot analysis and immunohistochemistry. The antibody recognized a band of approximately 65 kDa in Western blots using epididymal lysates. Immunohistochemical studies demonstrate that OCTN2 is present in the basolateral membrane of epithelial cells in the distal caput, corpus, and proximal cauda epididymides. In conclusion, OCTN2 is present in the rat epididymis in a region-dependent manner and is likely to be responsible for the transport of l-carnitine into the cells of the epididymal epithelium.