Thymosin beta 4 (T beta 4), a 43-residue peptide of uncertain function, is widely distributed in most tissues of vertebrates. Southern blot analysis demonstrated that the rat genome contains many closely related T beta 4 sequences. Two distinct T beta 4 sequences were isolated by screening a rat genomic DNA library. Analysis of these clones revealed that they represent T beta 4 retroposons; they lack introns and contain stretches of poly(A) sequence at their 3' ends and direct repeats flanking the cDNA-like sequences. In order to isolate a T beta 4 intron-containing gene, DNA fragments that include T beta 4 introns were generated by polymerase chain reaction, using rat genomic DNA as template. Subsequent Southern blot analysis of rat genomic DNA with intron-specific probes demonstrated the presence of a single T beta 4 intron-containing gene. Upon screening the rat genomic DNA library with an intron-specific probe, two overlapping clones of the T beta 4 intron-containing gene were isolated. Further characterization and sequence analysis showed that the rat T beta 4 intron-containing gene has a 2-kilobase pair-long transcription unit containing two introns. The transcription initiation site and putative regulatory elements of the promoter were identified.