In a cross between two isolates of the rice blast fungus Magnaporthe grisea, a spontaneous mutant was identified which was non-pathogenic on rice and did not differentiate appressoria on hydrophobic surfaces. The mutant was also non-pathogenic on rice when inoculated on wounded leaves, suggesting that its ability to grow in plant tissue was also impaired. Mutant spores were three-celled, with apical cells longer than those of parental isolates. Using one full sib-cross and one back-cross, we showed that all of the mutant phenotypes (non-pathogenicity on rice, appressorium deficiency and elongated spores) co-segregated as a single gene which was named APF1. Comparison of apf1- mutants with previously characterized appressorium-deficient mutants of M. grisea provided evidence that APF1 is a new gene involved in appressorium differentiation which has pleiotropic effects on plant colonisation and spore morphology. Significantly, apflmutants could not form appressoria in the presence of cyclic AMP, indicating that APF1 acts downstream, or independently, of the cAMP signalling pathway required for appressorium differentiation and function in M. grisea.