The sulfoconjugated steroids estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were separated in the reversed phase mode on polyamide-coated TLC plates. Baseline resolution was obtained between tritiated ES and DS standards when run with a mobile phase of 20% acetonitrile in 5mM aqueous triethylamine, triethanolamine, tris-hydroxymethylaminomethane, tributylamine or ammonia. ES and DS showed no mobility in the absence of an ion-pair reagent. The radioactive peaks were detected and integrated non-destructively by scanning. Quantitation was confirmed by elution of cut-out peak areas and liquid scintillation counting. Similar results were obtained with washed ethanol extracts of serum labeled with tritiated ES and DS. The extracts were defatted on the plate with hexane: ethyl acetate (1:1) prior to the reversed phase development.