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Objectives To examine the predictive relationships of early infant and caregiver variables on expressed pain-related negative affect duration at the 12-month immunization. Methods Infants and their caregivers (N = 255) were followed during immunization appointments over the first year of life. Latent growth curve modeling in a structural equation modeling context was used. Results Higher levels of initial infant pain reactivity at 2 months and caregiver emotional availability averaged across 2, 4, and 6 months of age were related to larger decreases in the duration of infant negative affect over the first 6 months of life. Longer duration of infant negative affect at 2 months and poorer regulation of infant negative affect over the first 6 months of life predicted longer durations of infant negative affect by 12 months. Conclusions Infant negative affect at 12 months was a function of both infant factors and the quality of caregiver interactive behaviors (emotional availability) in early infancy.