The fungal pathogen Neozygites floridana Weiser and Muma has been evaluated as a classical biological candidate for introduction into Africa against the invasive tomato red spider mite Tetranychus evansi Baker and Pritchard. In this study, the effect of temperature on sporulation, germination and virulence of three isolates of N. floridana collected from T. evansi in three climatically distinct regions of Brazil and Argentina was determined. Six constant temperatures of 13 degrees C, 17 degrees C, 21 degrees C, 25 degrees C, 29 degrees C and 33 degrees C were tested for their effect on the ability of the three fungal isolates to sporulate, germinate and kill the mites. Six alternating-temperature regimes of 17-13 degrees C, 21-13 degrees C, 29-13 degrees C, 33-13 degrees C, 33-23 degrees C, 33-28 degrees C under a 12h photophase were also tested to estimate virulence of the three isolates against T. evansi. The Vipos isolate discharged more conidia than isolates from Recife or Piracicaba at all temperatures and sporulation was strongly temperature dependent. Optimal sporulation rates were observed at 25 degrees C while optimal germination rates were observed at 25 degrees C and 29 degrees C. At 29 degrees C, the shortest mean survival time of T. evansi (3.16 days, 95% CI of 3.05-3.27) was observed for the isolate from Vipos, while the longest LT(50) (3.47 days, 95% CI 3.34-3.59) was observed for the isolate from Piracicaba. Mortality of mites increased as the differences between alternating day and night temperatures increased from 8 degrees C (21-13 degrees C), to 10 degrees C (33-23 degrees C), to 16 degrees C (29-13 degrees C), with smallest and highest temperature differences of 4 degrees C (17-13 degrees C) and 20 degrees C (33-13 degrees C), both producing low mortalities. The overall results suggest that the Vipos isolate is better adapted to a wider range of temperatures than the other isolates tested.