Escherichia coli strains associated with diarrhoeal disease have been classified into several types according to the pathogenic mechanism. Among these, enteroaggregative E. coli strains (EAggEC) have been associated with persistent childhood diarrhoea. Some strains of EAggEC produce a heat-stable toxin (EAST-1) that differs from others described previously. The main goal of this case-control study was to determine the prevalence of EAggEC and EAST-1-producing E. coli strains as a cause of diarrhoea in children in Spain and to study their in-vitro susceptibility to 21 antimicrobial agents. In the case group (115 children) 22 (19%) isolates and four (3.5%) isolates were EAST-1-producing E. coli and EAggEC, respectively, whereas in the control group (79 children) four (5%) isolates produced EAST-1 (p = 0.005) and three (3.8%) isolates were EAggEC. The present study suggests that EAST-1-producing E. coli strains are associated with diarrhoeal diseases in Spanish children, whereas EAggEC strains are not. Moreover, EAST-1-producing E. coli strains showed a high susceptibility to all the antimicrobial agents tested except for ampicillin.