Using a sample of 249 newborn Peruvian infants we investigated the stability and factorial structure of neonatal temperament, assessed in two different settings during the first week of life. While neonates were still in the hospital their behavior during a structured anthropometry examination was videotaped and subsequently coded on four temperament dimensions: activity level, negative emotionality, alertness and soothability. The same dimensions were coded using a videotape obtained during a subsequent visit to the neonates home. Results indicated that while there was modest stability of temperament across the two settings levels of neonatal temperament also varied across settings, with increased distress and reduced soothability in the more stressful laboratory examination and increased levels of alertness during the unstructured home observation. Across both settings neonatal temperament was defined by a primary component linking alertness and soothability with a reverse loading for irritability. We interpret this component as an early precursor of self-regulation. However, our results indicate that the structure of neonatal temperament also is sensitive to setting, with the second identified component linking activity level to irritability in the laboratory assessment and with alertness in the home observation. Results are discussed with reference both to current theories of the development of temperament and to the importance of assessment of contextual characteristics when assessing temperament, even in the neonatal period. © 2004 Elsevier Inc. All rights reserved.