Abstract Background The evaluation of infant meconium as a cumulative matrix of prenatal toxicant exposure requires comparison to established biomarkers of prenatal exposure. Methods We calculated the frequency of detection and concentration of tobacco smoke metabolites measured in meconium (nicotine, cotinine, and trans-3'-hydroxycotinine concentrations) and three serial serum cotinine concentrations taken during the latter two-thirds of pregnancy among 337 mother-infant dyads. We estimated the duration and intensity of prenatal tobacco smoke exposure using serial serum cotinine concentrations and calculated geometric mean meconium tobacco smoke metabolite concentrations according to prenatal exposure. We also compared the estimated associations between these prenatal biomarkers and infant birth weight using linear regression. Results We detected nicotine (80%), cotinine (69%), and trans-3'-hydroxycotinine (57%) in most meconium samples. Meconium tobacco smoke metabolite concentrations were positively associated with serum cotinine concentrations and increased with the number of serum cotinine measurements consistent with secondhand or active tobacco smoke exposure. Like serum cotinine, meconium tobacco smoke metabolites were inversely associated with birth weight. Conclusions Meconium is a useful biological matrix for measuring prenatal tobacco smoke exposure and could be used in epidemiological studies that enroll women and infants at birth. Meconium holds promise as a biological matrix for measuring the intensity and duration of environmental toxicant exposure and future studies should validate the utility of meconium using other environmental toxicants.