BACKGROUND: Although nurse clinicians and researchers use infant behaviors to indicate the responses of preterm infant to stimulation, little is known about how the biological factors of development, sleeping and waking states, infant characteristics, and infant illness severity affect preterm infant behaviors. OBJECTIVE: This study examined the development of eight infant behaviors over the preterm period and determined the relation of these behaviors to sleeping and waking and to infant characteristics and illness severity. METHODS: Seventy-one preterm infants were observed once per week from 7:00 p.m. to 11:00 p.m. from the time they were no longer critical until term or discharge. The occurrence of four sleep-wake states and eight behaviors were recorded every 10 seconds during the observations. RESULTS: Negative facial expressions increased over the preterm period; sighs, startle/jerks, jitters, and the likelihood of having hiccups decreased. Infant characteristics had only minor effects: boys had more negative facial expressions, and longer mechanical ventilation was associated with more sighs and jitters. All behaviors showed state-related differences in frequency. In addition, only startle/jerks and jitters showed the same developmental patterns within each state. CONCLUSIONS: Significant development of infant behaviors occurs over the preterm period but involves changes not only in the absolute percentage of each behavior but also in the percentages within each sleeping and waking state. Thus, preterm infant behaviors cannot be used clinically for assessment without consideration of the state in which they occur.