Yeasts growing limited for nitrogen source or treated with fusel alcohols form elongated cells--pseudohyphae. Absence of mitochondrial DNA or anaerobic conditions inhibits this process, but the precise role of mitochondria is not clear. We found that a significant percentage of pseudohyphal cells contained mitochondria with different levels of membrane potential within one cell. An uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), but not the ATP-synthase inhibitor oligomycin D, prevented pseudohyphal growth. Interestingly, repression of the MIH1 gene encoding phosphatase activator of the G2/M transition partially restores the ability of yeast to form pseudohyphal cells in the presence of FCCP or in the absence of mitochondrial DNA. At the same time, retrograde signaling (the one triggered by dysfunctional mitochondria) appeared to be a positive regulator of butanol-induced pseudohyphae formation: the deletion of any of the retrograde signaling genes (RTG1, RTG2, or RTG3) partially suppressed pseudohyphal growth. Together, our data suggest that two subpopulations of mitochondria are required for filamentous growth: one with high and another with low transmembrane potential. These mitochondria-activated signaling pathways appear to converge at Mih1p level.