The "pipette" or "growth cone turning" assay is widely used for studying how axons respond to diffusible guidance cues in their environment. However, little quantitative analysis has been presented of the gradient shapes produced by this assay, or how they depend on parameters of the assay. Here we used confocal microscopy of fluorescent gradients to characterize these shapes in 3 dimensions. We found that the shape, and more specifically the concentration at the position usually occupied by the growth cone in this assay, varied in sometimes unexpected ways with the molecular weight of the diffusible factor, charge, pulse duration and pulse frequency. These results suggest that direct observation of the gradient of the particular guidance factor under consideration may be necessary to quantitatively determine the signal to which the growth cone is responding.