Light-triggered gene expression systems offer an unprecedented spatiotemporal resolution that cannot be achieved with classical chemically inducible genetic tools. Here we describe a protocol for red light-responsive gene expression in mammalian cells. This system can be toggled between stable ON and OFF states by short pulses of red and far-red light, respectively. In the protocol, CHO-K1 cells are transfected to allow red light-inducible expression of the secreted alkaline phosphatase (SEAP) reporter, and gene expression is tuned by illumination with light of increasing wavelengths. As a starting point for elaborate red light-responsive gene expression, we outline the reversible activation of gene expression and describe how a spatial pattern can be 'printed' on a monolayer of cells by using a photomask. The core protocol requires only 4 d from seeding of the cells to reporter quantification, and other than light-emitting diode (LED) illumination boxes no elaborate equipment is required. © 2014 Nature America, Inc.