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HIV-1 infection in children less than 15 years of age is mainly due to mother-to-child transmission. The aim of this work was to investigate molecular evidence to prove father-to-be horizontal transmission in two possible events of transmission. In the first event a boy was identified as HIV infected at 2-3 years of age. At the same time infection was confirmed in the father, while mother and siblings were negative. In the second event a girl was negative for HIV at age 1 and identified as HIV-1 infected at age 6. The father's HIV infection was diagnosed in the same period while the mother was repeatedly negative. No evidence of sexual assault or transfusion was recorded in any case. Peripheral blood mononuclear cells were obtained from both fathers and children. After PCR amplification, the C2V3 region of the envelope gene and the region coding for amino acid 132 of p24 up to amino acid 40 of p7 of the gag gene were sequenced. Genetic distance measurements and phylogenetic tree analysis showed that in both cases the father's and child's viral sequences were closely related. They were distinct when compared to Argentina sequences including sequences from the same geographic region. Epidemiological and molecular data strongly suggest that horizontal transmission had occurred, probably related to the close father-to-child contact.