Sphingomonas melonis TY utilizes nicotine as a sole source of carbon, nitrogen, and energy to grow. One of the genes in its ndp catabolic cluster, ndpT, encodes a hypothetical transporter. Since no transporter for nicotine has been identified in microorganisms, we investigated whether NdpT is responsible for nicotine transport. ndpT was induced by nicotine, and gene knockout and complementation studies clearly indicated that ndpT is essential for the catabolism of nicotine in strain TY. NdpT-GFP was located at the periphery of the cells, suggesting that NdpT is a membrane protein. Uptake assays with L-[C-14] nicotine illustrated that nicotine uptake in strain TY is mediated by a constitutively synthesized permease with a K-m of 0.362 +/- 0.07M and a V-max of 0.762 +/- 0.068molmin(-1) (mg cell dry weight)(-1) and that ndpT may play a role in nicotine exclusion. Hence, we consider NdpT a nicotine catabolism-related protein.