Construction of a DNA plasmid that expresses a diphtheria toxin A chain (DT-A) gene under control of human immunodeficiency virus (HIV-1) proteins Tat and Rev has been described. Here the generation of HeLa cell clones containing integrated, HIV-regulated DT-A sequences is reported. Five such clones were identified by their decreased expression of a luciferase reporter gene transiently cotransfected with Tat- and Rev-encoding plasmids. The decreased luciferase expression most probably was due to activation of the integrated DT-A gene because higher luciferase activity could be restored by introducing either DT antitoxin or a gene encoding a mutant, DT-resistant elongation factor 2 (the intracellular target for DT-A). Analysis by polymerase chain reaction (PCR) indicated that all clones expressed DT-A encoding RNA. The clones were then transfected with an HIV proviral clone and were tested for HIV production; all five clones demonstrated substantially impaired HIV production compared with parental HeLa cells, as shown by p24 assays of culture supernatants. Our success in generating these cell lines indicates that extremely low basal expression has been achieved in view of the high cellular lethality of DT-A. HIV-regulated expression of DT-A may be applicable as a gene therapy approach for the acquired immune deficiency syndrome (AIDS), to bring about selective suicide of HIV-infected cells before production of viral progeny.