H1t is an H1 histone variant unique to late spermatocytes and early spermatids. Using gene targeting and embryonic stem cell technologies, we have produced mice with a disrupted H1t gene. Homozygous H1t-null mice have normal fertility and show no obvious phenotypic consequence due to the lack of this histone. Biochemical and immunohistochemical approaches were used to show that normal changes in chromosomal proteins occurred during spermatid development, including the appearance and disappearance of transition proteins 1 and 2. Both protamines 1 and 2 are present in normal amounts in sonication-resistant spermatid nuclei from H1t-null mice. Analysis of H1 histones by quantitative gel electrophoresis in enriched populations of pachytene spermatocytes and round spermatids showed that the lack of H1t is only partially compensated for by somatic H1s, so that the chromatin of these cells is H1 deficient. Because H1t is thought to create a less tightly compacted chromatin environment, it may be that H1-deficient chromatin is functionally similar to chromatin with H1t present, at least with respect to permitting spermatogenesis to proceed.