Faecal swabs obtained from a random sample of 268 cows and 90 calves on 19 Lugo (northwestern Spain) farms were examined for necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2). We found NTEC CNF1+ and CNF2+ on 11% and 95% of the farms, respectively, NTEC producing CNF2 were significantly more frequently isolated from calves (58%) than from cows (17%) (P < 0.001). The proportion of animals colonized with CNF2+ strains on each farm ranged from 0% to 60%. NTEC strains producing CNF2 isolated from healthy cattle belonged to 27 O serogroups; however, 64% were of one of 12 serogroups (O2, O8, O8-O75, O14, O15, O55, O86, O88, O115, O121, O147, and O168). Furthermore, the serogroups determined in CNF2+ strains isolated from cows (O2, O8, and O14) were different from those found in NTEC producing CNF2 isolated from calves (O8-O75, O15, O55, O86, O88, O115 and O147).