TFIIAα/β-like factor (ALF) is a testis-specific counterpart of the large subunit of human general transcription factor TFIIA. Northern analysis shows that ALF mRNA first appears in mouse testis at Postnatal Day 14. Similarly, expression of the general transcription factors TBP, TRF2, TFIIAα/β, TFIIAγ, and TFIIIB₉₀ is also increased beginning at Postnatal Day 14, suggesting that there is a coordinated induction of many general transcription factors during male germ cell differentiation. Analysis of male germ cells separated by Staput sedimentation shows that ALF is present in pachytene spermatocytes and haploid spermatids. In addition, in situ hybridization experiments with adult mouse testis shows that ALF is present in haploid spermatids. Searches of the human genome sequence database using the basic local alignment search tool reveal that the ALF and TFIIAα/β (GTF2A1) genes are both composed of nine exons, whereas the TFIIAγ (GTF2A2) gene is composed of five exons. Furthermore, nucleotide and amino acid comparisons among human and mouse ALF, TFIIAα/β, and TFIIAγ cDNA sequences show that ALF has diverged more rapidly than either TFIIAα/β or TFIIAγ. Finally, the ALF and SBLF (Stoned B-Like Factor) sequences present in the chimeric SALF cDNA are both present on human chromosome 2, and an analysis of the corresponding genes suggests a model for the formation of SALF.