Conidial germination of Colletotrichum trifolii was affected by concentrations of Tween 20 (polyoxyethylene sorbitan monolaurate). The highest rate of germination and appressorium formation was obtained in 0.083% Tween 20.Appressorium formation was more sensitive to temperature than was germination. The percentage of germinated spores which formed an appressorium was reduced when the spore suspensions were incubated for 24 h at incubation temperatures above 27 °C. Appressorium formation also was reduced when conidia were exposed, in culture or in suspension, to 35 °C for short periods. Similarly, germination was reduced with heat exposure, but at longer exposures than those necessary to reduce appressorium formation. Spores in suspension responded to a shorter heat exposure (4 h) than spores in culture.The reduced atmospheric CO2 level did not affect the amount of germination or appressorium formation. However, cell walls of appressoria produced in an atmosphere low in CO2 were thin-walled and without the brown pigment characteristic of normal appressoria.Protein synthesis inhibitors (cycloheximide and p-fluorophenylalanine) blocked both germination and appressorium formation. Nucleic acid synthesis inhibitors were either partially effective or ineffective and their mode of action in C. trifolii is unknown.