Induction of the cytoplasmic petite mutation in yeast by 5-fluorouracil (5FU) and 5-fluorocytosine (5FC) is known to depend on the incorporation of 5FU into some species of RNA; 5FC is active only following deamination to 5FU. Several mutants ahve now been isolated which are resistant to petite mutagenesis by 5FU but remain sensitive to growth inhibition by this analogue. They fall into two classes: those in class I are also resistant to mutagenesis by 5FC, while class II mutants retain partial sensitivity to the latter agent. The growth of both classes is sensitive to 5FC. The behavior of class II mutants requires that exogenous 5FU is specifically excluded from the site of synthesis of the target RNA involved in petite mutagenesis, while 5FC has access to it. The most likely explanation is that the RNA concerned is synthesized in the mitochondria, and that the mitochondrial membranes of class II mutants are impermeable to 5FU but not 5FC. This is supported by the finding that the membrane-active agent dimethylsulfoxide restored 5FU sensitivity to this class of mutants. No such effect was observed with class I mutants, and these are thought to have altered mitochondrial RNA-synthesizing systems which are unable to recognize fluorinated nucleotides.