Background: Acetaminophen (APAP) is the most commonly used medication during pregnancy. Increasingly, studies have reported potential adverse associations between maternal prenatal use of APAP and offspring asthma, adverse reproductive outcomes, and neurodevelopmental disorders. However, most studies have relied on self-reported maternal exposure which has the potential for exposure misclassifications. We conducted this study to compare maternal self-reported APAP intake and three acetaminophen metabolites measured in urine collected during pregnancy and umbilical cord serum samples. Methods We used maternal interview data and biospecimens from the Nutrition in Pregnancy cohort of 2291 pregnant women recruited from Connecticut and Western Massachusetts during 1996-2000. We selected three exposure groups including 10 mothers who reported frequent intake (14 days+) of APAP in the first 3 months of pregnancy, 10 who reported frequent intake in the last 3 months of pregnancy, and 10 who reported never using APAP in these two periods from the cohort. We analyzed three APAP metabolites (parent compound, acetaminophen glucuronide, and 3-(N-acetyl-l-cystein-S-yl)-acetaminophen) levels in the maternal urine (avg. 24 gestational weeks) and the umbilical cord serum samples. Results The parent APAP compound and 3-(N-acetyl-l-cystein-S-yl)-acetaminophen) were detected in all urine and serum samples analyzed (100%), while acetaminophen glucuronide was less detectable in the cord serum (40% vs. 100% in urine). APAP metabolites in maternal urine and cord serum were correlated (r=0.43-0.70) within mother-child pairs. All three urinary and cord serum APAP metabolite levels were more strongly correlated with the reported number of days of APAP intake in late pregnancy (r=0.57-0.73; p-values <0.03, rank tests), and less with early pregnancy intake (r=0.18-0.34; p-values 0.21-0.60, rank tests). Conclusions Our findings suggest detailed intake data collected in maternal interviews is an efficient and valid measure to indicate exposure to APAP in pregnancy, while biomarker measures can be used to represent a specific exposure period of interest.