Ras and Rho GTPases have been examined in a wide variety of eukaryotes and play varied and often overlapping roles in cell polarization and development. Studies in Saccharomyces cerevisiae and mammalian cells have defined some of the central activities of these GTPases. However, these paradigms do not explain the role of these proteins in all eukaryotes. Unlike yeast, but like more complex eukaryotes, filamentous fungi have Rac-like proteins in addition to Ras and Cdc42. To investigate the unique functions of these proteins and determine how they interact to co-ordinately regulate morphogenesis during growth and development we undertook a genetic analysis of GTPase function by generating double mutants of the Rho GTPases cflA and cflB and the newly isolated Ras GTPase rasA from the dimorphic pathogenic fungus, Penicillium marneffei. P. marneffei growth at 25 degrees C is as multinucleate, septate, branched hyphae which are capable of undergoing asexual development (conidiation), while at 37 degrees C, uninucleate pathogenic yeast cells which divide by fission are produced. Here we show that RasA (Ras) acts upstream of CflA (Cdc42) to regulate germination of spores and polarized growth of both hyphal and yeast cells, while also exhibiting CflA-independent activities. CflA (Cdc42) and CflB (Rac) co-ordinately control hyphal cell polarization despite also having unique roles in regulating conidial germination and polarized growth of yeast cells (CflA) and polarized growth of conidiophore cell types and hyphal branching (CflB).