This chapter provides methods and insights into the use of antisense RNA as a molecular genetic tool. Posttranscriptional inhibition of specific gene expression can be achieved by antisense RNA fragments under control of a conditional promoter. Effective titration of gene expression can cause an apparent null mutation or can be modulated to levels of interest in comparison with wild type. Validation of antisense RNA can be achieved by both RNA and protein quantitation techniques. Applications include phenotypic studies of genes in response to specific stimuli, environments, or the contribution of genes in regulatory networks. This chapter will focus on shotgun-cloned antisense for comprehensive gene identification and cell-based hypersensitivity assays for antibiotic screening. Antisense RNA strategies have high utility when the target gene is essential for survival and needs to be compared with wild type.