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Despite the availability of efficacious prevention of mother-to-child transmission (PMTCT) interventions and improved access to preventive services in many developing countries, vertical HIV transmission persists. A matched case–control study of HIV-exposed infants between January and June 2012 was conducted at 20 clinics in Kenya. Cases were HIV-infected infants and controls were exposed, uninfected infants. Conditional logistic regression analysis was conducted to determine characteristics associated with HIV infection. Forty-five cases and 45 controls were compared. Characteristics associated with HIV-infection included poor PMTCT service uptake such as late infant enrollment (odds ratio [OR]: 7.1, 95% confidence interval [CI]: 2.6–16.7) and poor adherence to infant prophylaxis (OR: 8.3, 95%CI: 3.2–21.4). Maternal characteristics associated with MTCT included lack of awareness of HIV status (OR: 5.6, 95%CI: 2.2–14.5), failure to access antiretroviral prophylaxis (OR: 22.2, 95%CI: 5.8–84.6), and poor adherence (OR: 8.1, 95%CI: 3.7–17.8). Lack of clinic-based HIV education (OR: 7.7, 95%CI: 2.0–25.0) and counseling (OR: 8.3, 95%CI: 2.2–33.3) were reported by mothers of cases. Poor uptake of PMTCT services and a reported absence of HIV education and counseling at the clinic were associated with MTCT. More emphasis on high-quality, comprehensive PMTCT service provision are urgently needed to minimize HIV transmission to children.