Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod outer segments (ROS) is a key amplification step in phototransduction. Definitive estimates of the turnover number, k(cat), and of the K(m) are crucial to quantifying the amplification contributed by the PDE. Published estimates for these kinetic parameters vary widely; moreover, light-dependent changes in the K(m) of PDE have been reported. The experiments and analyses reported here account for most observed variations in apparent K(m), and they lead to definitive estimates of the intrinsic kinetic parameters in amphibian rods. We first obtained a new and highly accurate estimate of the ratio of holo- PDE to rhodopsin in the amphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose structural integrity was systematically varied. In the most severely disrupted ROS preparation, we found K(m) = 95 μM and k(cat) = 4,400 cGMP · s-1. In suspensions of disc-stack fragments of greater integrity, the apparent K(m) increased to ~600 μM, though k(cat) remained unchanged. In contrast, the K(m) for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated apparent K(m) of suspensions of disc stacks can be explained as a consequence of diffusion with hydrolysis in the disc stack, which causes active PDEs nearer the center of the stack to be exposed to a lower concentration of cyclic GMP than PDEs at the disc stack rim. The analysis predicts our observation that the apparent K(m) for cGMP is elevated with no accompanying decrease in k(cat). The analysis also predicts the lack of a K(m) shift for cAMP and the previously reported light dependence of the apparent K(m) for cGMP. We conclude that the intrinsic kinetic parameters of the PDE do not vary with light or structural integrity, and are those of the most severely disrupted disc stacks.