The instability of the shear layer separated from a circular cylinder was investigated for Reynolds numbers, based on cylinder diameter, from 3500 to 53 000. For this, the most amplified instability frequencies were found and compared against available results for this ReD range. Based on this initial set of measurements, ReD=3500 was chosen for a detailed study of the instability of the separated shear layer to 3-D disturbances. This involved the excitation of plane Kelvin–Helmholtz waves using an acoustic source, and pairs of oblique waves of equal but opposite angles produced by roughness which was distributed evenly along the cylinder span at the separation line. The sensitivity of the shear layer instability to the initial 2-D disturbance amplitude and 3-D disturbance wave angle was documented. The results showed that there exists a most amplified 3-D wave angle (spanwise wave length) for the separated shear layer. The sensitivity to the wave angle agreed well with the linear 3-D stability analysis of a Stuart vortex by Pierrehumbert and Widnall and the numerical simulations of a free shear flow by Rogers and Moser.