The flow over a backward-facing step with laminar separation was investigated experimentally under controlled perturbation for a Reynolds number of 11 000, based on a step height h and a free-stream velocity U0. The reattaching shear layer was found to have two distinct modes of instability: the 'shear layer mode' of instability at St-theta almost-equal-to 0.012 (St-theta = f-theta/U0, theta being the momentum thickness at separation and f the natural roll-up frequency of the shear layer); and the 'step mode' of instability at St(h) almost-equal-to 0.185 (St(h) = fh/U0). The shear layer instability frequency reduced to the step mode one via one or more stages of a vortex merging process. The perturbation increased the shear layer growth rate and the turbulence intensity and decreased the reattachment length compared to the unperturbed flow. Cross-stream measurements of the amplitudes of the perturbed frequency and its harmonics suggested the splitting of the shear layer. Flow visualization confirmed the shear layer splitting and showed the existence of a low-frequency flapping of the shear layer.