The goal of this study was to determine the period prevalence of mother-child separation (i.e., living apart from mother) and factors associated with separation between women infected with HIV type 1 and their children (17 years old or younger). A retrospective cohort study was conducted, based on routinely collected data by the Family Advocacy, Care and Education Services (FACES) of Children's Hospital (New Orleans, Louisiana) on children (n ؍ 586) of 231 HIV-infected mothers followed from July 1, 2001 to March 20, 2003. The 3-month period prevalence of separation from mother ranged between 12.7% and 21.2% over the course of follow-up. At baseline, 14% of children were separated from their mothers. Over the course of follow-up, older children (6 to 17 years), those with mothers who used illegal drugs and/or alcohol and who suffered from mental health problem(s), those whose mothers did not live at a permanent residence, and children with mothers who had a CD4 cell count below 200 cells per microliter were more likely to be separated from their mother. No significant differences in substance use, mental health, CD4 count, or other sociodemographics were identified between women lost to follow-up and those who remained in care. Substance use and mental health problems played a role in maternal-child separation among the HIV-positive women in this sample, even after clinical status and socioeconomic factors were taken into account. Providing additional resources for substance use and mental health treatment may help to prevent or reduce maternal-child separation among similar populations. 509