It was first shown by W.L. Russell (1962), and confirmed by him and others, that a 24-h interval between dose fractions (but not shorter or longer ones) elevates the rate of radiation-induced spermatogonial specific-locus mutations to levels considerably above the linear extrapolation made from lower-dose results. We have now analyzed the nature of mutations induced either in previously undisturbed or in "sensitized" spermatogonial stem cells, i.e., those that received a challenging dose of X-rays 24 h following a priming dose. Results are based on molecular studies of a large set of viable albino mutations [using probes derived from the tyrosinase (c) gene and from the regions surrounding c], and on retrospective classifications of mutations at c and two additional loci into LL (large lesions), IG (intragenic mutations), and OL (other lesions), utilizing criteria developed earlier. A significant difference (P = 0.016) was found between previously undisturbed and sensitized stem-cell spermatogonia; the latter have a higher LL/IG ratio, similar to the ratio observed for mutations induced in poststem-cell stages. This finding of a qualitative difference indicates that the additional mutations produced by a 24-h fractionated treatment are the result of the second (challenging) dose. The qualitative difference, further, indicates that the mutation-rate-augmenting effects of 24-h fractionation are not due, merely, to an increase (caused by the priming dose) of a normally responsive component of the spermatogonial population. The finding that the additional mutations that are produced by the challenging dose are primarily large DNA lesions suggests that the nuclear state of sensitized stem-cell spermatogonia may be different from the state of previously undisturbed spermatogonia. This state, which appears to be similar to that of postspermatonial stages, may be conducive to the formation of LLs, even by agents that are not LL inducers in other systems. The results further indicate that the relative paucity of LLs characteristic of treated (previously undisturbed) spermatogonial stem cells is probably not the result of selection against such mutations during subsequent germ-cell development.