Assessment and management of pain in preterm infants is critical and complicated. The addition of salivary cortisol measurement may improve the specificity of assessment and guide care to alleviate pain. The purpose of this study was fourfold: (a) assess the feasibility of a method of saliva collection in premature infants, (b) assess reliability of a method of measuring salivary cortisol in response to heelstick, (c) identify relationships between salivary cortisol and a measure of pain behavior (using CRIES) following heelstick, and (d) identify peak response times for elevations of salivary cortisol following heelstick in preterm infants. This was a prospective, descriptive pilot study. Serial saliva samples were collected from eight healthy infants 30 to 36 weeks' gestational age in a Newborn Intensive Care Unit. Cortisol levels were determined using enzyme-immune assay. Samples were collected without use of stimulants. Sample means supported peak and trough patterns previously described in the literature. Behavioral measures of pain did not correlate well with peak cortisol levels.