Single-locus (sl), multiplex (m), and semi-nested (sn) polymerase chain reaction (PCR) procedures were developed for the detection of Yersinia enterocolitica and Aeromonas hydrophila in raw milk samples. Two oligonucleotide primers for each pathogen were used in PCR, to detect the yst gene of Y, enterocolitica and aer gene of A. hydrophila. The amplified fragment size was 145 base-pair (bp) for yst gene, and 209 bp for the aer gene. The performance of the systems were evaluated with seeded milk samples and naturally contaminated raw milk samples. PCR results were compared with conventional cultural procedures. The limits of detection of sIPCR and mPCR assays were approximately 10(2) cfu ml(-1) (0.5 cfu/PCR reaction mixture) for both pathogens in seeded raw milk. When studied with naturally contaminated raw milk samples, detection rates obtained by PCR and cultural methods were 53% and 36% for Y. enterocolitica and were 23% and 14% for A. hydrophila, respectively These results indicate that the direct PCR analysis of raw milk can be used as a rapid and specific diagnostic method for both pathogens. (C) 2000 Academic Press.