Experiments were conducted in the field and laboratory to determine effects of low temperatures on Xylella fastidiosa populations in American sycamore. Roots and shoots from naturally infected trees at two locations were collected monthly. Sap extracted from the samples was tested by enzyme-linked immunosorbent assay for presence of X. fastidosa and was diluted and plated on periwinkle wilt medium to determine populations of viable bacteria. Cumulative rainfall and hours below temperature thresholds (-5 to 10degreesC) were recorded at each site. Bacterial populations in shoots were negatively correlated with cumulative hours below -5degreesC air temperature (r = -0.96). In roots, bacterial populations were only weakly correlated with cumulative hours below soil temperature thresholds (-0.61 < r < -0.25). Bacterial populations were not correlated with monthly rainfall. In the laboratory, resistant and susceptible sycamore trees were inoculated with X. fastidiosa and held in the dark at 5degreesC or 22degreesC. After 12 weeks, inoculated stem sections were collected and sap was extracted and tested as described previously. Stems that tested positive for X. fastidiosa were divided into additional samples and tested as described above. Results of the laboratory study indicated no significant effects of low-temperature treatment (5degreesC) or host resistance on viable bacteria. Bacterial detection frequency and population size were greatest near the inoculation point and the primary direction of early bacterial spread was acropetal.