Noise in the rod photoreceptors limits the ability of the dark-adapted visual system to detect dim lights. We investigated the molecular mechanism of the continuous component of the electrical dark noise in toad rods. Membrane current was recorded from intact, isolated rods or truncated, internally dialyzed rod outer segments. The continuous noise was separated from noise due to thermal activation of rhodopsin and to transitions in the cGMP-activated channels. Selectively disabling different elements of the phototransduction cascade allowed examination of their contributions to the continuous noise. These experiments indicate that the noise is generated by spontaneous activation of cGMP phosphodiesterase (PDE) through a process that does not involve transducin. The addition of recombinant gamma, the inhibitory subunit of PDE, did not suppress the noise, indicating that endogenous gamma does not completely dissociate from the catalytic subunit of PDE during spontaneous activation. Quantitative analysis of the noise provided estimates of the rate constants for spontaneous PDE activation and deactivation and the catalytic activity of a single PDE molecule in situ.