Over time, organisms have taken different paths to complexity. Although only a single eukaryotic cell, Tetrahymena thermophila derives complexity from its remarkably complex cell surface architecture. Proteomic analysis from our earlier experiments has led to the identification of nearly four dozen proteins isolated from the cortical cytoskeleton. A high molecular coiled coil structural protein (Epc1), an EF‐hand calcium binding protein (Tcb2), and a protein kinase (Epk1) have been investigated. These proteins are believed to have crucial regulatory and structural functions in addition to playing a role in calcium‐dependent regulation of ciliary motion. We are investigating the interactions of these proteins during cell division through confocal microcopy, fluorescence spectroscopy, and NMR solution studies. It was evident through the utilization of confocal microscopy that these proteins were found to localize to a narrow layer of the cortical cytoskeleton called the epiplasm, which was consistent with previous purification experiments. Fluorescence spectroscopy was used in order to monitor conformational changes in the C‐terminal domain of Tcb2, and we identified the presence of one high affinity calcium‐binding site with a low micromolar dissociation constant. The shift peaks observed during (1)H‐(15)N HSQC‐NMR monitored calcium titrations of the Tcb2 C‐terminal domain corroborated evidence of calcium‐dependent conformational changes in this domain. NMR spectra also illustrated that in both the presence and absence of calcium this domain is well folded and upon calcium addition undergoes dramatic conformational change. In the future, we will explore protein distributions relative to one another through FRET and double‐labeling experiments.