The bacteriophage T4 nrdB gene, encoding nucleoside diphosphate reductase subunit B, contains a self-splicing group I intervening sequence. The nrdB intron was shown to be absent from the genomes of the closely related T-even phages T2 and T6. Evidence for variable intron distribution was provided by autocatalytic 32P-guanosine 5'-triphosphate labeling of T-even RNAs, DNA and RNA hybridization analyses, and DNA sequencing studies. The results indicate the nonessential nature of the intron in nrdB expression and phage viability. Furthermore, they suggest that either precise intron loss from T2 and T6 or lateral intron acquisition by T4 occurred since the evolution of these phages from a common ancestor. Intron movement in the course of T-even phage divergence raises provocative questions about the origin of these self-splicing elements in prokaryotes.