Because human toxaphene exposure data are largely lacking, we surveyed human serum pools collected from U.S. residents to determine the feasibility of measuring toxaphene in human samples and to determine whether additional analytical requirements were needed for routine measurement of toxaphene. We report a method for quantification of toxaphene congeners in human serum using a mixed-bed gradient solid-phase extraction and analysis using gas chromatography-high-resolution mass spectrometry with electron-impact ionization. In this method, we monitored low-mass fragment ions that were common to all 22 congeners. To verify the specific congeners detected, we further analyzed the extract using negative methane chemical ionization. We used this method to measure two specific congeners, Parlar 26 and 50, at concentrations ranging from about 3 to 30 pg/ml (0.7-7 ng/g lipid) in pooled human serum collected in Atlanta, Chicago, and Cincinnati. We identified several analytical parameters that must be strengthened to routinely measure toxaphene congeners in human samples.