There is currently no published report on the isolation and definitive identification of histone H1 in Saccharomyces cerevisiae. It was, however, recently shown that the yeast HHO1 gene codes for a predicted protein homologous to H1 of higher eukaryotes (Landsman, D. (1996) Trends Biochem. Sci. 21, 287-288; Ushinsky, S. C., Bussey, H. , Ahmed, A. A., Wang, Y., Friesen, J., Williams, B. A., and Storms, R. K. (1997) Yeast 13, 151-161), although there is no biochemical evidence that shows that Hho1p is, indeed, yeast histone H1. We showed that purified recombinant Hho1p (rHho1p) has electrophoretic and chromatographic properties similar to linker histones. The protein forms a stable ternary complex with a reconstituted core di-nucleosome in vitro at molar rHho1p:core ratios up to 1. Reconstitution of rHho1p with H1-stripped chromatin confers a kinetic pause at approximately 168 base pairs in the micrococcal nuclease digestion pattern of the chromatin. These results strongly suggest that Hho1p is a bona fide linker histone. We deleted the HHO1 gene and showed that the strain is viable and has no growth or mating defects. Hho1p is not required for telomeric silencing, basal transcriptional repression, or efficient sporulation. Unlike core histone mutations, a hho1Delta strain does not exhibit a Sin or Spt phenotype. The absence of Hho1p does not lead to a change in the nucleosome repeat length of bulk chromatin nor to differences in the in vivo micrococcal nuclease cleavage sites in individual genes as detected by primer extension mapping.