We have initiated a study of ancient male migrations from Siberia to the Americas using Y chromosome polymorphisms. The first polymorphism examined, a C-->T transition at nucleotide position 181 of the DYS199 locus, was previously reported only in Native American populations. To investigate the origin of this DYS199 polymorphism, we screened Y chromosomes from a number of Siberian, Asian, and Native American populations for this and other markers. This survey detected the T allele in all five Native American populations studied at an average frequency of 61%, and in two of nine native Siberian populations, the Siberian Eskimo (21%) and the Chukchi (17%). This finding suggested that the DYS199 T allele may have originated in Beringia and was then spread throughout the New World by the founding populations of the major subgroups of modern Native Americans. We further characterized Native American Y chromosome variation by analyzing two additional Y chromosome polymorphisms, the DYS287 Y Alu polymorphic (YAP) element insertion and a YAP-associated A-->G transition at DYS271, both commonly found in Africans. We found neither African allele associated with the DYS199 T allele in any of the Native American or native Siberian populations. However, we did find DYS287 YAP+ individuals who harbored the DYS199 C allele in one Native American population, the Mixe, and in one Asian group, the Tibetans. A correlation of these Y chromosome alleles in Native Americans with those of the DYS1 locus, as detected by the p49a/p49f (p49a,f) probes on TaqI-digested genomic DNA, revealed a complete association of DYS1 alleles (p49a,f haplotypes) 13, 18, 66, 67 and 69 with the DYS199 T allele, while DYS1 alleles 8 and 63 were associated with both the DYS199 C and T allele.