Rat testis contains a unique H1 histone variant (H1t) not detected in a variety of other rat organs. H1t is extracted from chromatin by salt as expected for an H1 component (released by 0.6 M NaCl but not by 0.3 M); however, in contrast to other mammalian H1 species, H1t is not extracted in significant amounts by 5% (w/v) trichloroacetic acid. H1t is undetectable in testes from 3-or 15-day-old rats, but is evident in extracts from 21-day-old animals. Its initial appearance in the developing testis thus correlates with the progression of germinal cells to the meiotic stage of differentiation. H1t was isolated in homogeneous form by extraction of whole tissue with 0.75 M perchloric acid, fractional precipitation with trichloroacetic acid, and two cycles of chromatography over a cation exchange resin (Bio Rex 70). The amino acid composition of H1t differs significantly from that of somatic type H1 variants (for example, higher arginine and methionine content). Its identification as an H1 species is supported by analysis of the two fragments resulting from treatment of the protein with N-bromosuccinimide. H1t, as well as the six recognized somatic type H1 components of rat testis may be resolved by two-dimensional polyacrylamide gel electrophoresis run in the absence of denaturants at pH 4.5 in the first dimension and in the presence of sodium dodecyl sulfate in the second dimension.