We describe a method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability. This method utilizes a chloramphenicol-resistant plasmid containing a temperature-sensitive (ts) replication origin. In this method, the target gene is first cloned into the ts vector and the recombinant plasmid is used to transform wild-type B. subtilis. The second step involves transformation of the resulting strain with a linear DNA fragment containing a second antibiotic resistance marker (tet) that disrupts the gene of interest. Selection for tetracycline resistance forces a double crossover between the chromosomal and fragment-borne copies of the gene, thereby replacing the wild-type gene in the chromosome with the disrupted allele. Cells survive even if the gene is essential by virtue of the complementing plasmid. Transformants are then grown at the non-permissive temperature for plasmid replication under various growth conditions. Isolation of chloramphenicol-sensitive colonies indicates that the gene is dispensable, whereas the inability to isolate chloramphenicol-sensitive colonies indicates that the gene is essential. The general utility of this method is demonstrated by allowing disruption of mtrA and trpE under conditions that render each gene non-essential, but not under growth conditions in which each gene is essential.