Prohibitins are ubiquitous, abundant proteins found in a wide range of organisms and that have a high degree of sequence conservation. In yeast it has previously been demonstrated that prohibitin proteins form a complex and are involved in maintaining the morphological and functional integrity of mitochondria. We have used a colony-sectoring assay as a screen for mutants that are dependent upon the presence of functional Phb2p in the cell. Two classes of prohibitin dependent mutation (pbd1 and pbd2) were isolated and characterised. The effect of these mutations on replicative lifespan was determined, demonstrating that the pbd1 mutant slightly extended lifespan, whereas in contrast, the pbd2 mutation resulted in a shortening in both the mean- and the maximum-lifespan. The pbd1 mutation was also found to reduce chronological lifespan. Reducing the expression of the PHB2 gene in the pbd mutants was found to retard the rate of growth and to affect replicative lifespan. As the two mutants behave in a different manner they probably affect different aspects of prohibitin function.