Plasmid-encoded toxin (Pet) is a heat-labile enterotoxin encoded in the enteroaggregative Escherichia coli (EAEC) virulence plasmid. Several evidence support the role of this 108-kDa secreted protein in the pathogenesis of EAEC diarrhea. In this study, we standardized a slot blot immunoassay for Pet detection. EAEC culture supernatants were applied onto a polyvinylidene difluoride membrane, and, using rabbit polyclonal Pet antisera, the expression of the toxin by slot blot immunoassay was observed in 9.5% of the isolates studied. In addition, no negative control reacted with Pet antiserum in this assay. This assay is a rapid, specific, reproducible, and low-cost methodology, therefore demonstrating its potential in diagnosing Pet expression. Moreover, we describe for the first time that expression of Pet can be directly detected from EAEC culture supernatants and may be used in clinical laboratorial routine instead of polymerase chain reaction detection of the pet gene, especially in developing countries where the EAEC pathotype has been considered an emerging pathogen.