The rat interleukin 4 (IL 4) gene has been isolated from a genomic lambda phage library by cross-hybridization to the mouse IL 4 cDNA. Like the mouse and human counterparts, it exists as a single copy gene in the genome and consists of four exons. The overall structure of the IL 4 locus seems highly conserved. This is indicated by the low degree of restriction fragment length polymorphism in a number of laboratory and wild mice and by the conservation of the intron size between human, rat, and mouse IL 4 genes. Furthermore, evolutionary conserved elements are the promoter region, the position of cysteine residues and sequence motifs in the 3' untranslated regions that are believed to be involved in destabilization of the mRNA. In contrast, the predicted amino acid sequence of the rat IL 4 gene shows low homology (57%) with the mouse homologue. The divergence between mouse and rat IL 4 genes is even more pronounced in the carboxy-terminal region (47% homology in the last 68 amino acids). The ratio between replacement and silent mutations in the IL 4 genes of different species suggests a complex pattern of selective forces acting on the IL 4 gene, which includes both selection against and for amino acid substitutions in individual positions. The functional identity with IL 4 has been confirmed by expression of the gene and the demonstration of the ability to induce MHC class II antigen expression on spleen cells.