Leaf spot and stem canker caused by Sphaerulina vaccinii is associated with premature defoliation in lowbush blueberry resulting in reduced yields. In this study, we investigated the impact of free water, RH, temperature, light, and plant age on leaf infection under controlled conditions. On potato dextrose agar, germination of conidia was usually polar. Growth was minimal at 5 and 10°C, increased at 15 and 20°C, was maximal at 25°C and decreased at 30°C. Percentage of germinated conidia on inoculated blueberry leaves incubated in dark controlled-humidity chambers for 3 days (25°C) was 86.0, 90.5, 81.3, and 28.3 in free water, 100, 97.5 or 95% RH, respectively. Germination did not occur at 90 or 85% RH. Infection of inoculated plants, however, was not favored by free water, but rather by high RH (>95%) and a 14-h photoperiod (180 μmol/m2 per second). Infection failed in continuous darkness, continuous light, or continuous darkness followed by 4, 8, or 12 h of light. Light and scanning electron microscopy showed that hyphal penetration into stomata on abaxial leaf surfaces was strongly tropic. When germ tubes grew in close proximity to a stomate, a penetration hypha formed at ∼90° angles to the germ tube and took the closest path to the stomate. Stomatal penetration was usually direct, but occasionally appressorium-like hyphal swellings formed over stomatal openings. When inoculated plants were exposed to high RH (>95%) at various temperatures, infection occurred after 4 days at 10°C, after 3 days at 15°C and after 1 day at 20 and 25°C. Infection failed to occur at 30°C. Disease severity also increased with duration of the humid period. When leaves were examined microscopically, those that had been incubated for 6 days showed a substantially greater network of epiphytic growth with more stomatal penetrations compared with those incubated for 3 days. Infection was substantially reduced when the humid period was interrupted by alternating days of low RH (60%). Two-week-old leaves were 2.7 times more susceptible than 8-week-old leaves.