Tritium beta radiation (3H beta-radiation) in the form of tritiated water was used to induce mutations at the alcohol dehydrogenase (Adh) locus in male Drosophila melanogaster post-meiotic germ cells. All 23 Adh null mutations were large deletions (> 20 kb), determined by genetic complementation and Southern blot analyses. 27 Adh null mutations have been induced by 100-kVp X-rays (Aaron, 1979) and have been genetically and molecularly characterized (Ashburner et al., 1982; Chia et al., 1985; LoMonaco et al., 1987; Mahmoud et al., 1991). In contrast to 3H beta-radiation, 100-kVp X-rays induced a bimodal distribution of Adh null mutations, intragenic mutations, < or = 250 bp, and large deletions, > 100 kb. A statistically significant difference was observed between the frequency of large deletions (23/23 or 1.0) induced by 3H beta-radiation and the frequency of large deletions (19/27 or 0.7) induced by 100-kVp X-rays. However, a statistical difference was not observed between the size distribution of the large deletions induced by 3H beta-radiation and X-rays. The relative deletion frequency (RDF) induced by 3H beta-radiation and 100-kVp X-rays was (1.0/0.7 = 1.4). The relative biological effectiveness (RBE) of these two radiation sources was 1.4, determined from the ratio of the regression coefficients of the respective 3H beta-radiation and X-ray sex-linked recessive lethal (SLRL) dose-response data. The large difference in size between the two classes of X-ray-induced Adh null mutations and the increase in mutation frequency and deletion frequency for 3H beta-radiation with respect to X-rays may indicate that the relative deletion frequency (RDF) is the molecular biological basis for the increase in the RBE for radiation sources with a mean LET value < or = 10 keV/microns.