Human somatic cell variants resistant to the arginine analog, canavanine, express 200-fold increased levels of argininosuccinate synthetase (AS) mRNA as compared to parental cells. In this study we examined whether AS cDNA sequences contain cis-acting regulatory elements that are involved in the induction of AS mRNA in canavanine-resistant cells. Minigene constructs containing AS cDNA sequences under the transcriptional control of a viral promoter were stably transfected into the human squamous cell carcinoma line, RPMI 2650. Upon conversion of cells to canavanine-resistance, expression of the endogenous AS gene increased by two orders of magnitude as expected. Surprisingly, however, expression of AS cDNA minigenes decreased 10- to 15-fold in canavanine-resistant cell variants. The observed down-modulation of AS cDNA minigene expression was dependent upon a concomitant induction of the endogenous AS gene and not simply expression of the canavanine-resistant phenotype. This paradoxical regulation was specific for AS gene sequences since a minigene containing the neomycin-resistance gene in place of AS cDNA sequences failed to regulate. Furthermore, minigenes lacking a substantial portion of the AS cDNA also failed to exhibit the down-modulation. These findings suggest that expression of the human AS gene is regulated by a specific and limiting, positively-acting, trans-acting mechanism in canavanine-resistant cells and that exogenous AS cDNA (mRNA) sequences can compete for this mechanism.