Seven mutations that yield thermolabile DNA polymerases have been isolated in the DNA polymerase I gene, POL1, of Saccharomyces cerevisiae. Strains carrying the mutant genes are temperature sensitive for growth. The pol1 mutants were identified by a method that has general applicability for identification of both temperature-sensitive and null mutations. A plasmid containing a mutagenized pol1 gene was transformed into a strain in which the only functional copy of the POL1 gene was carried on an unstable plasmid. The genes conferring temperature-sensitive growth were detected after elimination of the unstable plasmid containing the wild-type gene. DNA polymerase I isolated from each of the mutants is defective at both 23 degrees C and 36 degrees C. DNA synthesis is deficient in vivo at 36 degrees C in all the mutants, while RNA synthesis is normal in all but one of the mutants. The terminal phenotype of pol1 temperature-sensitive mutants is dumbbell-shaped cells in which the nucleus has migrated to, but apparently not entered, the isthmus separating the mother and the daughter. The POL1 gene is located on chromosome XIV approximately 2 centimorgans away from met4.