Because of their prominent roles in regulation of gene expression, it is important to understand how levels of Krupple-like transcription factors SP1 and SP3 change in germ cells during spermatogenesis. Using immunological techniques, we found that both factors decreased sharply during meiosis. SP3 declined during the leptotene-to-pachytene transition, whereas SP1 fell somewhat later, as spermatocytes progressed beyond the early pachytene stage. SP3 reappeared for a period in round spermatids. For Sp1, the transition to the pachytene stage is accompanied by loss of the normal, 8.2-kb mRNA and appearance of a prevalent, 8.8-kb variant, which has not been well characterized. We have now shown that this pachytene-specific transcript contains a long, unspliced sequence from the first intron and that this sequence inhibits expression of a reporter, probably because of its many short open-reading frames. A second testis-specific Sp1 transcript in spermatids of 2.4 kb also has been reported previously. Like the 8.8-kb variant, it is compromised translationally. We have confirmed by Northern blotting that the 8.8-, 8.2-, and 2.4-kb variants account for the major testis Sp1 transcripts. Thus, the unexpected decline of SP1 protein in the face of continuing Sp1 transcription is explained, in large part, by poor translation of both novel testis transcripts. As part of this work, we also identified five additional, minor SpI cap sites by 5' rapid amplification of cDNA ends, including a trans-spliced RNA originating from the Glcci1 gene.