Polyfluoroalkyl chemicals (PFCs) comprise a group of man-made organic compounds, some of which are persistent contaminants with developmental toxicity shown in laboratory animals. There is a paucity of human perinatal exposure data. The US EPA conducted a pilot study (Methods Advancement for Milk Analysis) including 34 breastfeeding women in North Carolina. Milk and serum samples were collected at 2-7 weeks and 3-4 months postpartum; 9 PFCs were assessed in milk and 7 in serum. Perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS) were found in nearly 100% of the serum samples. PFOS and PFOA were found at the highest concentrations. PFCs were below the limit of quantification in most milk samples. Serum concentrations of PFOS, PFOA and PFHxS were lower (p<0.01) at the second visit compared to the first visit. Living in North Carolina 10 years or longer was related to elevated PFOS, PFOA and PFNA (p<or=0.03). These pilot data support the need to further explore perinatal PFC exposures and potentially related health effects, as planned in the upcoming National Children's Study which provided the framework for this investigation.