<jats:title>Abstract</jats:title><jats:p>Three strains of rats were fed a fish oil diet to verify their ability to incorporate and convert dietary eicosapentaenoic acid (20∶5ω3) into trienoic prostaglandins. Our results show that such conversion indeed occurs in kidney medullae homogenates. Specifically, the presence of prostaglandin E<jats:sub>3</jats:sub> (PGE<jats:sub>3</jats:sub>) was established by gas chromatographic‐mass spectrometric (GC‐MS) analysis. That compound was conclusively identified by comparison of fragment ions and their relative intensities with those obtained from authentic PGE<jats:sub>3</jats:sub>. Further evidence was provided by studying the recovery of exogenously added PGE<jats:sub>3</jats:sub>. The crude ethyl acetate extracts of the medullary homogenates were methylated and cleaned up by liquid‐gel chromatography with Lipidex‐5000 prior to conversion to PGB<jats:sub>3</jats:sub> for GC‐MS analysis. The PGE<jats:sub>3</jats:sub> was quantified by selected ion monitoring (SIM) with [3,3,4,4‐<jats:sup>2</jats:sup>H<jats:sub>4</jats:sub>PGE<jats:sub>2</jats:sub> as internal standard. The levels of PGE<jats:sub>3</jats:sub> were similar, about 3 ng/mg of wet tissue, in the 3 strains of rats. Identical in vivo conversion of the 20∶5ω3 fatty acid to PGE<jats:sub>3</jats:sub> could not be positively established by analysis of pooled urine specimens.