Functional characterization of adenylyl cyclase (AC) isoforms has proven challenging in mammalian cells because of the endogenous expression of multiple AC isoforms and the high background cAMP levels induced by nonselective AC activators. To simplify the characterization of individual transmembrane AC (mAC) isoforms， we generated a human embryonic kidney cell line 293 (HEK293) with low cAMP levels by knocking out two highly expressed ACs， AC3 and AC6， using CRISPR/Cas9 technology. Stable HEK293 cell lines lacking either AC6 (HEK-ACΔ6) or both AC3 and AC6 (HEK-ACΔ3/6) were generated. Knockout was confirmed genetically and by comparing cAMP responses of the knockout cells to the parental cell line. HEK-ACΔ6 and HEK-ACΔ3/6 cells revealed an 85% and 95% reduction in the forskolin-stimulated cAMP response， respectively. Forskolin- and G-coupled receptor-induced activation was examined for the nine recombinant mAC isoforms in the HEK-ACΔ3/6 cells. Forskolin-mediated cAMP accumulation for AC1-6 and AC8 revealed 10- to 250-fold increases over the basal cAMP levels. All nine mAC isoforms， except AC8， also exhibited significantly higher cAMP levels than the control cells after G-coupled receptor activation. Isoform-specific AC regulation by protein kinases and Ca/calmodulin was also recapitulated in the knockout cells. Furthermore， the utility of the HEK-ACΔ3/6 cell line was demonstrated by characterizing the activity of novel AC1 forskolin binding-site mutants. Hence， we have developed a HEK293 cell line deficient of endogenous AC3 and AC6 with low cAMP background levels for studies of cAMP signaling and AC isoform regulation.