The aim of this study was to differentiate S-cone responses from other retinal activities using various recording conditions and to optimize these recording conditions for clinical diagnostics. S-cone responses to blue stimuli (449 nm) were studied in 20 healthy subjects and four patients with enhanced S-cone syndrome. The time-integrated luminance of the stimulus varied from 0.008 to 1.0 cd s/m2. Three isoluminant backgrounds were used (100 ph cd/m2=40 sc cd/m2): amber (594 nm), green (513 nm), and red (635 nm). With low flash strengths (from 0.008 to 0.032 cd s/m2), the S-cone response appeared as a single positive peak, while with higher strengths (≥0.064 cd s/m2), it appeared as a second peak that followed the L-cone and M-cone components. With a further increase in flash strength (≥0.25 cd s/m2), the S-cone response interfered with the i-wave of the L-cone and M-cone systems. The wavelength and luminance of the background influenced the suppression of the rods, as well as the L-cone- and M-cone-system activities. The S-cone response was measurable in the presence of the amber and green backgrounds, but its amplitude was higher if a strong red background was used. Thus, the function of the retinal S-cone system can be measured if possible interference from other retinal sources can be minimized by the appropriate combination of stimulus and background parameters.