This study aimed to analyze factors associated with delayed initiation of breastfeeding (lack of breastfeeding in the first hour of life). This was a cross-sectional study conducted in 2009 with 673 postpartum women at hospitals belonging to the Brazilian Unified National Health System (SUS) in the city of Rio de Janeiro, Brazil. Statistical analysis used multilevel logistic regression, with two levels (individual and hospital). Prevalence of delayed initiation of breastfeeding was 49.2%. Delivery in a Baby-Friendly Hospital (BFH) had a protective effect against late initiation of breastfeeding (OR = 0.17; 95%CI: 0.05-0.55), while cesarean section (OR = 5.95; 95%CI: 3.88-9.12) and ignorance of HIV status until delivery (OR = 2.16; 95%CI: 1.04-4.50) increased the odds of delay. Reduction in cesarean rates, adherence to protocols to prevent vertical HIV transmission, and expanded accreditation of hospitals in the BFH are important strategies to promote breastfeeding in the first hour of life.