Ingested ethanol is transported to the colon via blood circulation, and intracolonic ethanol levels are equal to those of the blood ethanol levels. In the large intestine, ethanol is oxidized by colonic bacteria, and this can lead to extraordinarily high acetaldehyde levels that might be responsible, in part, for ethanol‐associated carcinogenicity and gastrointestinal symptoms. It is believed that bacterial acetaldehyde formation is mediated via microbial alcohol dehydrogenases (ADHs). However, almost all cytochrome‐containing aerobic and facultative anaerobic bacteria possess catalase activity, and catalase can, in the presence of hydrogen peroxide (H2O2, use several alcohols (e.g., ethanol) as substrates and convert them to their corresponding aldehydes. In this study we demonstrate acetaldehyde production from ethanol in vitro by colonic contents in a reaction catalyzed by both bacterial ADH and catalase. The amount of acetaldehyde produced by the human colonic contents was proportional to the ethanol concentration, the amount of colonic contents, and the length of incubation time, even in the absence of added nicotinamide adenine dinucleotide or H2O2. The catalase inhibitors sodium azide and 3‐amino‐1,2,4‐triazole (3‐AT) markedly reduced the amount of acetaldehyde produced from 22 mM ethanol in a concentration dependent manner compared with the control samples (0.1 mM sodium azide to 73% and 10 mM 3‐AT to 67% of control). H202 generating system [β‐D(+)‐glucose + glucose oxidase] and nicotinamide adenine dinucleotide induced acetaldehyde formation up to 6‐ and 5‐fold, respectively, and together these increased acetaldehyde formation up to 11‐fold. The mean supernatant catalase activity was 0.53 ± 0.1 jumol/min/mg protein after the addition of 10 mM H202, and there was a significant (p < 0.05) correlation between catalase activity and acetaldehyde production after the addition of the hydrogen peroxide generating system. Our results demonstrate that colonic contents possess catalase activity, which probably is of bacterial origin, and indicate that in addition to ADH, part of the acetaldehyde produced in the large intestine during ethanol metabolism can be catalase dependent.