Although research has demonstrated that the neonatal intensive care unit (NICU) is a stressful environment for preterm and high-risk infants, little research validates the suspected relationships between infant biobehavioral responses and environmental stress in the NICU. This exploratory study examined the relationship between environmental stress and biobehavioral responses of preterm infants. The study used a repeated-measures research design to examine research variables in one group of preterm infants. Measurements of research variables were recorded every 2 minutes during two 60-minute observation periods for each research day (1 hour in the morning and 1 hour in the afternoon) and conducted over 2 days. A convenience sample of 37 preterm infants was recruited from 2 medical centers in Taiwan. A total of 4164 observations were made and recorded during the study. There was a statistically significant (P < .05) relationship between environmental stressors and changes in physiological signals. There were also statistically significant (P < .05) relationships between environmental stress and some specific stress behaviors. This research is applicable to neonatal clinical practice because it demonstrates the importance of recognizing the preterm infant's biological stress responses to environmental stressors, allowing for early interventions to reduce the possibility of more serious physiological or pathological changes in the status of the preterm infant.