PDZK1 (also known as CAP70, NHERF3, or NaPi-Cap1) is a scaffolding protein composed of four PDZ (Post-Synaptic Density-95, Discs Large, Zonula Occludens-1) domains followed by a short carboxyl-terminal tail. This scaffold acts as a mediator of localization and expression levels of multiple receptors in the kidney, liver, and endothelium. Here, we characterize the self-association properties of the protein. PDZK1 can undergo modest homodimerization in vivo and in vitro through self-association involving its third PDZ domain. In addition, the tail of PDZK1 interacts in an intramolecular fashion with the first PDZ domain, but this interaction does not contribute to dimer formation. The interaction between the tail of PDZK1 and its first PDZ domain induces the protein to adopt a more compact conformation. A head-to-tail association has also been reported for EBP50/NHERF1, a two-PDZ domain member of the same scaffolding protein family as PDZK1, and shown to regulate binding of target proteins to the EBP50 PDZ domains. As opposed to EBP50, the association of PDZK1 with specific ligands for its PDZ domains is unaffected by the intramolecular association, establishing a different mode of interaction among these two members of the same scaffolding family. However, the tail of PDZK1 interacts with the PDZ domains of EBP50, and this interaction is negatively regulated by the intramolecular association of PDZK1. Thus, we have uncovered a regulated association between the two PDZ-containing scaffolding molecules, PDZK1 and EBP50.