Dietary polyunsaturated fatty acids (PUFA) reduce colonic proliferation and exert a mild laxative effect. We have studied the effect of the highly unsaturated eicosapentaenoic acid ethyl ester (EPA-EE) on the growth and metabolism of colonic bacteria in vitro, and in vivo. For the in vitro study, growth was assessed by viable counts. Bacteroides thetaiotaomicron was significantly inhibited in anaerobic media containing EPA-EE at concentrations > 7 g/l. Escherichia coli was apparently resistant even at 100 g/l. For the in vivo study, ten healthy volunteers ingested 18 g EPA-EE/d for 7 d, Stool frequency, 24 h stool weight and whole-gut transit time were assessed together with breath H-2 and (CO2)-C-14 excretion following oral ingestion of 15 g lactitol labelled with 0.18 MBq [C-14]lactitol. The area under the breath-H-2-time curve was significantly reduced by EPA-EE, from a control value of 690.3 (SE 94.2) ppm.h to 449.5 (SE 91.7) ppm.h. Percentage dose of (CO2)-C-14 excreted, total stool weight and whole-gut transit time were unaltered, being respectively 24 (SE 2)%, 281 (SE 66) g and 45 (SE 4) h with EPA-EE v. control values of 27 (SE 1)%, 300 (SE 89)g and 42 (SE 5) h. It is concluded that dietary supplementation with EPA-EE reduces breath H-2 excretion without apparently impairing overall colonic carbohydrate fermentation. The observed reduction may reflect utilization of H-2 to hydrogenate the five double bonds of EPA-EE.