AbstractGene expression circuitries with time‐delayed expression profiles regulate key events, such as oscillating systems, noise elimination, and coordinated multi‐step processes, in all organisms from bacteria to mammalian cells. We present the rational synthesis of a genetic circuit displaying time‐delayed expression in silico and in mammalian cells. The network is based on a time‐delay circuit, where the tetracycline‐responsive transactivator (tTA) induces expression of the pristinamycin‐responsive repressor PIP‐KRAB, which silences expression of the terminal human placental secreted alkaline phosphatase (SEAP). While the addition of pristinamycin I inactivates PIP‐KRAB and results in the immediate resumption of SEAP expression, addition of tetracycline abolishes PIP‐KRAB synthesis. Consequently, SEAP production remains repressed until the PIP‐KRAB buffer in the cell is eliminated. We characterized in silico and in vivo the time‐delayed expression properties and analyzed the impact of the size and stability of the PIP‐KRAB buffer on fine‐tuning of the response kinetics. This tunable time‐delay circuitry represents a biologic building block for emulating a fundamental circuit topology in integrated artificial synthetic gene networks for the design of tailor‐made cell types and organisms. Biotechnol. Bioeng. 2007;98: 894–902. © 2007 Wiley Periodicals, Inc.