Father-infant and mother-infant (one-year-olds) adrenocortical attunement was explored during the Strange Situation Procedure (SSP) among 125 father-infant and 141 mother-infant dyads. Cortisol was assessed at baseline (T1), 20 (T2), and 40 minutes (T3) after the first parent-infant separation. Initial correlations indicated significant associations between father-infant and mother-infant cortisol at each time. Cortisol interdependence was further explored using Actor-Partner Interdependence Models. There was no evidence supporting cortisol interdependence based on within-time residual correlations between parent-infant cortisol, once stability and cross-lagged paths were controlled. Infant cortisol at T2 predicted T3 cortisol for fathers and mothers resulting in a series of follow-up exploratory analyses to examine mediating processes which revealed that infant distress during the SSP predicted infant T2 cortisol, which, in turn, predicted infant negativity during the 15-min mother-infant teaching task that followed the SSP. Among father-infant dyads, infant T2 cortisol predicted infant negativity during father-infant interaction, with infants expressing more negativity having less sensitive fathers. Findings provide little support of parent-infant adrenocortical attunement across either father-infant or mother-infant dyads during the SSP, but preliminary evidence indicates infant distress as a potential mediator. Future research may want to focus on affective and behavioral processes that underlie the concept of parent-infant adrenocortical attunement.