Deactivation of the vertebrate photopigment rhodopsin is achieved through a two-step process. Rhodopsin is first phosphorylated by rhodopsin kinase and subsequently deactivated by the binding of the regulatory protein arrestin or its splice variant, p44. Although much is known about the overall differences between arrestin and p44 binding to different rhodopsin species (photolyzed versus unphotolyzed and/or phosphorylated versus unphosphorylated), the exact role of p44 during phototransduction remains to be fully elucidated. Our current study addresses this question by identifying structural differences between arrestin and p44 and characterizing the interaction between the negatively charged rhodopsin tail and either p44 or arrestin. Our results demonstrate that arrestin and p44 bind differently to different phosphorylated rhodopsin species and that this may be due to a structural difference between p44's and arrestin's basal states. This difference offers a potential regulatory mechanism that could regulate p44 and arrestin binding and, as a result, regulate the kinetics of the rod's light response.