A high serum concentration of enterolactone, an enterolignan produced by colonic microbiota from precursors in cereals, vegetables, and fruits, is associated with reduced risk of acute coronary events. Probiotics and prebiotics modify colonic metabolism and may affect the serum enterolactone concentration. The effects of a probiotic mixture alone and with galactooligosaccharides (GOS) on serum enterolactone concentration and fecal metabolism were investigated in 18 healthy men. Participants received 3 interventions, each for 2 wk: 1) probiotics [Lactobacillus rhamnosus strains GG (LGG) and LC705, Propionibacterium freudenreichii ssp. shermanii JS, and Bifidobacterium breve Bb99, for a total amount of 2 × 10(10) CFU/d]; 2) probiotics and GOS 3.8 g/d; 3) probiotics, GOS, and rye bread (minimum 120 g/d). Serum enterolactone and fecal dry weight, enzyme activities, pH, SCFA, lactic acid bacteria, bifidobacteria, propionibacteria, and the strains LGG and LC705 were determined. The serum enterolactone concentration (nmol/L) tended to be decreased from baseline [mean (95% CI) 18.6 (10.8-26.4)] by probiotics alone [15.2 (7.8-22.7); P = 0.095], was not significantly affected by probiotics with GOS [21.5 (13.2-29.8)], and was increased by probiotics with GOS and rye bread [24.6 (15.4-33.7); P < 0.05]. Probiotics alone did not affect fecal β-glucosidase activity and bifidobacteria, but probiotics with GOS decreased β-glucosidase activity and increased bifidobacteria compared with baseline (P < 0.05) and with probiotics alone (P < 0.01). In conclusion, this probiotic mixture with or without GOS does not significantly affect serum enterolactone concentration. Because probiotics with GOS decreased fecal β-glucosidase activity but not serum enterolactone, the reduced fecal β-glucosidase, within the range of activities measured, does not seem to limit the formation of enterolactone.