Abstract Efforts to understand laser bioeffects in cells and tissues have been hindered by a non-uniform cellular response of the specimen resulting in graded biochemical effects. In addition, the small beam diameters of commonly used lasers limit the number of cells expressing a response to numbers inadequate for the study of biochemical effects. For a limited emission power, expansion of the beam diameter reduces the irradiance thus requiring longer exposure durations to produce a cellular response. Cultured human retinal epithelial cells were exposed as a single spot (tophat exposure) from a carbon dioxide (CO₂) laser operating at 10.6 µm or scanned with a raster system and compared to thermal injury produced with heated saline for short periods (1-9 s) at relatively high temperature (55-70 °C). Cell viability and induction of the heat shock response were evaluated. Initial attempts to use a tophat (uniform energy distribution) exposure resulted in a non-uniform cellular response (and non-uniform energy distribution) due to diffraction effects from the 2 mm selection aperture. However, raster scanning for appropriate times with the CO₂ laser yielded uniform cell viability and heat shock protein synthesis that were comparable to dipping cells in heated saline. The described scanning system produced a uniform cellular response that was correlated with the thermal injury model achieved by dipping of the cells in a heated saline bath. The scanning technique may also be applied to studies of cellular responses to other lasers to evaluate photochemical and photomechanical effects.