We have used fluorescent in situ hybridization to localize telomeres within the nuclei of sperm from six mammals (human, rat, mouse, stallion, boar, and bull). In minimally swollen sperm of mouse and rat, most of the telomeres are clustered within a limited area in the posterior part of nuclei. In sperm of other species, telomeres associate into tetrameres and dimers. On swelling of sperm cells with heparin/dithiotriethol, telomere associations disperse, and hybridization signals become smaller in size and their numbers approach or correspond to the number of chromosome ends in a haploid genome. Quantitation of telomere loci indicates that dimeric associations are prominent features of mammalian sperm nuclear architecture. Higher order telomere-telomere interactions and organization develop during meiotic stages of human spermatogenesis. At this stage, telomeres also become associated with the nuclear membrane. In an attempt to elucidate the molecular mechanisms underlying telomere interactions in sperm, we have identified a novel protein activity that binds to the double-stranded telomeric repeat (TTAGGG)n. Sperm telomere binding protein(s) (STBP) was extracted from human and bull sperm by 0.5 M NaCl. STBP does not bind single-stranded telomeric DNA and is highly specific for single base substitutions in a duplex DNA sequence. Depending on the conditions of binding, we observed the formation of several nucleoprotein complexes. We have shown that there is a transition between complexes, which indicates that the slower migrating complex is a multimer of the higher mobility one. We propose that STBP participates in association between the telomere domains which were microscopically observed in mammalian spermatozoa.