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Toxins produced by Penicillium species are reported in maize silage and have been associated with health problems in cattle. Our objectives were to evaluate the prevalence and dynamics of patulin (PAT), mycophenolic acid (MPA), cyclopiazonic acid (CPA), and roquefortine C (ROC) in fresh and ensiled maize. To achieve these objectives we developed a high-performance liquid chromatography method coupled with mass spectrometry to detect all four toxins simultaneously in silage. In addition we collected weather data, information on agronomic practices, and silage fermentation characteristics for each study site. Silage was collected at harvest and after ensiling in 2001 and 2002 from 30 Pennsylvania dairies. The average concentration of toxins (range in parentheses) was: PAT 0.08 microg/g (0.01 to 1.21), MPA 0.16 microg/g (0.02 to 1.30), CPA 0.12 microg/g (0.02 to 1.43), and ROC 0.38 microg/g (0.01 to 5.71). ROC was the most frequently detected toxin (60%), followed by MPA (42%), CPA (37%), and PAT (23%). Of 120 samples tested, 15% contained no detectible levels of toxin, 25% were contaminated with one toxin, 32% with two, 18% with three, and 10% with all four toxins. All four mycotoxins were found in freshly harvested material, contradicting the belief that Penicillium toxin formation occurs exclusively during storage. We observed that weather conditions during specific growth stages of the crop affected the final concentration of toxins in freshly harvested maize. In ensiled material, PAT levels were affected by concentrations of propionic and isobutyric acids. Based on our data, Penicillium mycotoxins can form while the crop is in the field and after ensiling, suggesting that preventative measures should begin prior to ensiling.