An improved specific analytical method for ochratoxin A (OTA) determination in olive oil is described, using a methanolic-aqueous extraction, an immunoaffinity column clean up step and high-pressure liquid chromatography with fluorescence detection. The mean recovery was found at 108% (relative standard deviation, RSD = 4.7%) and the detection limit (DL) was estimated at 4.6 ng kg(-1). Along with OTA, aflatoxin B(1) (AFB(1)) was determined using the same extract. The recovery factor was 84.8% (RSD = 17.8%) and the DL was 56 ng kg(-1) olive oil. Both determinations were applied in 50 samples of olive oil originated from representative regions of Greece. Results revealed the presence of OTA in 88% of samples tested (n = 44, mean 267 ng kg(-1)). Among them, 10 were contaminated with more than 500 ng kg(-1) (median 568 ng kg(-1)), 10 with 200-500 ng kg(-1) (median 260 ng kg(-1)), 15 with 100-200 ng kg(-1) (median 140 ng kg(-1)), nine with DL-100 (median 60 ng kg(-1)) and in six samples, OTA was not detectable. Interestingly, most contaminated samples were from Southern Greece. Results of AFB(1) determination showed the presence of aflatoxin B(1) (60 ng kg(-1)) in only one olive oil sample also from Southern Greece. The levels of OTA found in Greek olive oil were relatively low as compared with other commodities such as cereals or wine reported in the literature.