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To determine the acceptability and feasibility of universal HIV testing of 6-week-old infants attending immunization clinics to achieve early diagnosis of HIV and referral for HIV treatment and care services.An observational cohort with intervention.Routine HIV testing of infants was offered to all mothers bringing infants for immunizations at three clinics in KwaZulu Natal. Blood samples were collected by heel prick onto filter paper. Dried blood spots were tested for HIV antibodies and, if present, were tested for HIV DNA by PCR. Exit interviews were requested of all mothers irrespective of whether they had agreed to infant testing or not.Of 646 mothers bringing infants for immunizations, 584 (90.4%) agreed to HIV testing of their infant and 332 (56.8%) subsequently returned for results. Three hundred and thirty-two of 646 (51.4%) mothers and infants thereby had their HIV status confirmed or reaffirmed by the time the infant was 3 months of age. Overall, 247 of 584 (42.3%) infant dried blood spot samples had HIV antibodies indicating maternal HIV status. Of these, 54 (21.9%) samples were positive for HIV DNA by PCR. This equates to 9.2% (54/584) of all infants tested. The majority of mothers interviewed said they were comfortable with testing of their infant at immunization clinics and would recommend it to others.Screening of all infants at immunization clinics is acceptable and feasible as a means for early identification of HIV-infected infants and referral for antiretroviral therapy.