Virulent and weakly virulent grape strains of Xylella fastidiosa grew well on PD3 amended with gelatin and produced zones of hydrolysis on this medium, indicating the presence of proteolytic activity. Proteases also were produced in PD3 broth and degraded gelatin, azocasein, and azocoll. Gelatin was the best substrate for the proteases. In addition to using the gelatin assay, native activity and sodium dodecyl sulfate activity gels aided in visualizing and monitoring protease activity. Strain P of X. fastidiosa produced at least two proteases, designated P1 and P2, of 54 and 50 kDa, respectively. Protease activity was most abundant in the 31-60% ammonium sulfate fractions. Protease production was positively correlated with bacterial growth in PD3 broth and reached a maximum near the stationary phase of bacterial growth. P2 became more and PI became less predominant over time in PD3 broth. Protease activity was not diminished by temperatures up to 60 C and was optimal at 50 C and pH 9.0. P1 and P2 were similarly affected by pH and temperature.