[14C]-labelled thromboxane B2 and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-14C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E2, A2, D2 and F2alpha. Chromatographic properities in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-14C]-arachidonic acid to thromboxane B2 and prostaglandin E2 by human lung fibroblasts in tissue culture.