A series of human histone H1 subtype-specific antibodies are described that were generated for localization and functional studies. Since our previous attempts to produce such antibodies against intact subtypes met with limited success, resulting in one antibody against a subtype we have designated H1-3, the approach used in the work presented is based on the production of antibodies against synthetic peptides or peptide fragments encompassing the variant NH2-terminal region of each protein. Subtype-specific antibodies were obtained against synthetic peptides derived from subtypes designated H1-1 and H1-2 and the NH2-terminal fragment from an N-bromosuccinimide digest of H1-4. Antibody specificities were documented in all cases by enzyme-linked immunosorbent and protein immunoblot assays against the purified subtypes as well as immunoblots against whole cell and nuclear extracts. In addition, the in vivo distribution of each antibody was determined by indirect immunofluorescence. H1-1 appears to be distributed in parallel with DNA concentration, similar to the results with an antibody that recognizes all subtypes. However, H1-2 and H1-4 are non-uniformly distributed, exhibiting similar punctate staining patterns. The staining patterns described are different from the pattern described for the distribution of H1-3, suggesting that several subtypes are concentrated in distinct regions of the nucleus and, therefore, may be associated with distinct regions of the genome.