The mutagenicity of the trifunctional alkylating (or cross-linking) agent TEM (triethylenemelamine or 2,4,6-tris(1-aziridinyl)-1,3,5-triazine) in the adenine-3 (ad-3) region was studied with a two-component heterokaryon (H-12) of Neurospora crassa. The objective was to characterize the genetic damage produced by this chemical to determine the spectrum of specific-locus mutations induced in a lower eukaryotic organism and to compare this spectrum with that induced in the mouse. Specific-locus mutations in the ad-3 region of strain H-12 result from gene/point mutations, multiple-locus mutations, and multilocus deletion mutations at the closely linked ad-3A and ad-3B loci. These loci control two sequential biochemical reactions in the purine biosynthetic pathway. A 0.1 M solution of TEM was used to treat conidial suspensions of H-12 for 20, 40, 80, 120, or 170 min to obtain dose-response curves for (1) inactivation of conidia, and (2) the induction of specific-locus mutations in the ad-3 region. These experiments demonstrated that TEM is a strong mutagen (maximum forward-mutation frequency between 100 and 1000 ad-3 mutations per 10(6) survivors) for the induction of specific-locus mutations in the ad-3 region. Both biochemical and classical genetic tests were used to characterize the TEM-induced ad-3 mutations from each of the five treatment groups to distinguish between the different genotypic classes and subclasses. The overall data base from these genetic studies demonstrates that TEM-induced ad-3 mutations result predominantly (95.5% [769/805]) from gene/point mutations at the ad-3A and ad-3B loci, and from a low percentage (4.5% [36/805) of multilocus deletion mutations. In addition, TEM induces an unusually high frequency of multiple-locus mutations with sites of recessive lethal damage closely linked with the ad-3 region. Comparison of the dose-response curves for the major classes and subclasses of TEM-induced ad-3 mutations demonstrates (1) that gene/point mutations and multilocus deletion mutations increase as the 1.4 power of TEM treatment time, and (2) that the two classes of TEM-induced multiple-locus ad-3 mutations consisting of gene/point mutations with separate sites of recessive lethal damage increase at about the 1.96 power of TEM treatment time. When the data from the present specific-locus studies are compared with those in the mouse, we find, insofar as such comparisons are possible, that a similar spectrum of specific-locus mutations has been induced by TEM in each assay system.