We analysed the frequency of six Y-specific polymorphisms in 105 Amerindian males from seven different populations, 42 Caucasian males, and a small number of males of African, Chinese, and Melanesian origin. The combination of three of the six polymorphisms studied produced four different Y-haplogroups. The haplogroup A (non-variant) was the most frequent one. Eighty-five percent of Amerindians showing haplogroup A have the alphoid II (ahII) and the DYS19A Y-specific markers, an association that is found only in 10% of Caucasians and that has not been detected in Asiatics and Africans. Haplogroups C (YAP1) and D (YAP1 plus an A = G transition in the locus DYS271) are of African origin. Four percent of Amerindians and ,12% of Caucasians showed haplogroup C; ,1% of Amerindians and ,2% of Caucasians had haplogroup D. Haplogroup B is characterized by a C = T transition in nucleotide position 373 of the SRY gene domain; this haplogroup is found in Caucasians (,12%) and Amerindians (,4%). None of the Amerindians exhibiting the haplogroups B, C, or D show the haplotype ahII/ DYS19A. By haplotyping the Alu insert and the DNA region surrounding the insert in YAP1 individuals, we could demonstrate that Amerindian Y chromosomes bearing African markers (haplogroups C and D) are due to recent genetic admixture. Most non-ahII/DYS19A Amerindian Y-chromosomes in haplogroup A and most cases in haplogroup B are also due to gene flow. We show that haplotype ahII/DYS19A is in linkage disequilibrium with a C = T transition in the locus DYS199. Our results suggest that most Amerindian Y-chromosomes derive from a single paternal lineage characterized by the ahII/DYS19A/DYS199T Amerindian-specific haplotype. The analysis of a larger sample of native American Y-chromosomes will be required in order to confirm or correct this hypothesis.