We demonstrated that neonatal isolation (ISO) enhances cocaine self-administration in male and female adult rats and alters ventral striatal extracellular levels of serotonin (5-HT) and dopamine (DA) basally or in response to psychostimulants in infant rats. Now, we examine basal 5-HT, DA, and norepinephrine (NE) levels in nucleus accumbens (NAc) using in vivo microdialysis in adult male and female rats with or without ISO experience. NAc shows estrous cycle-dependent effects as do behavioral responses to cocaine. Because our prior work showed ISO eliminated estrous-cycle effects on behavior, we now test separate groups of females in proestrus, estrus, or diestrus stages. Litters were assigned to the ISO (1-h isolation; postnatal days 2-9) or non-handled (NH) condition. During adulthood (postnatal day 70-90), microdialysis probes were implanted and aimed at NAc core. Ten samples were collected over 150-min and measures of 5-HT, DA, and NE were analyzed via HPLC. ISO did not affect 5-HT levels in males. However, ISO modified estrous stage effects on 5-HT. The pattern of 5-HT levels in NH females (higher in diestrus and proestrus vs. estrus) was reversed in ISO females. DA levels were unaffected by ISO, similar to our findings at other ages, and did not differ by gender or estrous stage. None of these factors affected NE levels. Because 5-HT modulates DA and levels of both transmitters are increased by cocaine, this neurochemical effect of ISO may contribute to the ability of ISO to alter the behavioral responses to cocaine as we showed previously.