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To assess the characteristics and outcomes of antiretroviral treatment (ART) interruption (TI) in perinatally HIV-infected children.The Adolescent Master Protocol (AMP) of the Pediatric HIV/AIDS Cohort Study is a prospective cohort study that enrolled 7- to 16-year-old perinatally HIV-infected children between 2007 and 2009 from 15 sites in the United States and Puerto Rico.TI was defined as ART discontinuation for ≥3 months after ≥6 months of continuous ART. Subjects with and without TI were compared. Rates of change (slopes) in CD4+ T-lymphocyte (CD4) count and percentage (%) per month during TI were calculated. Factors related to CD4 slope in univariable analyses were included in multivariable linear regression.Of 444 eligible AMP subjects, 101 (23%) had at least one TI. Subjects with TI were born in earlier years but were otherwise similar to those without TI. For 81 TI subjects with complete data, the median (range) CD4% and CD4 count slopes were −0.66% per month (−3.54% to +1.34% per month) and −12.7 cells per cubic millimeter per month (−148 cells/mm3 to +31 cells/mm3 per month), respectively. On multivariable linear regression, there was a trend for lower CD4% slope to be associated (P < 0.1) with female sex, higher CD4% at TI, and higher peak viral load before TI. Advanced HIV disease stage and numerous ART regimens were more common in TI subjects in the lowest (fastest declining) CD4% slope quartile.TIs in perinatally HIV-infected youth are common. During TIs, CD4 values decline on average but with high intersubject variability. Factors predicting CD4 slope during TI need further study.