By a combination of monoclonal antibody (mAb)‐based enzyme‐linked immunosorbent assay (ELISA) and a simplified extraction procedure, the contents of ochratoxin A (OTA) in pig, cow and horse serum and in mixed feeds for pigs were analyzed. Preliminary ELISA with anti‐OTA mAb. 7/alkaline phosphatase (ALP)‐labelled second antibody has revealed that all 19 sera of pigs contained OTA at an average of 0–4 ngml‐1, and these data were closely correlated with HPLC analysis (r = 0.903). OTA in the sera was confirmed by both chemical methylation and enzymatic hydrolysis. The levels of OTA in one fetal pig serum sample and in five cow serum samples were below the detection limit of the ELISA (<0.1 ngml‐1). HPLC analysis revealed 0.3 and 0.7 μg kg‐1of OTA in two samples of mixed feeds. The second survey on 139 serum samples with an improved ELISA utilizing horseradish peroxidase (POD) as enzyme label giving a detection limit of 0.01 ngml‐1) has demonstrated average levels of 0.36, 0.02 and 0–11 ng ml‐1for OTA in serum from pig (124 samples), cow (14 samples) and horse (1 sample), respectively. The third survey on 17 pig sera also demonstrated that all samples were contaminated with OTA at an average of 5.2 ngml‐1. These three trials indicated that the present procedure based on the ELISA with anti‐OTA mAb. 7/POD‐labelled second antibody and the simple extraction procedure is an excellent tool for mass survey of OTA in livestock sera. This is the first report demonstrating the natural occurrence of OTA in livestock sera and mixed feeds in Japan.