The DUR3 gene, which encodes a component required for active transport of urea in Saccharomyces cerevisiae, has been isolated, and its sequence has been determined. The deduced DUR3 protein profile possesses alternating hydrophobic and hydrophilic regions characteristics of integral membrane proteins. Strong negative complementation observed during genetic analysis of the DUR3 locus suggests that the DUR3 product may polymerize to carry out its physiological function. Expression of DUR3 is regulated in a manner similar to that of other genes in the allantoin pathway. High-level expression is inducer dependent, requiring functional DAL81 and DAL82 genes. Maintenance of DUR3 mRNA at uninduced, nonrepressed basal levels requires the negatively acting DAL80 gene product. DUR3 expression is highly sensitive to nitrogen catabolite repression and also has a partial requirement for the GLN3 product.