Pinholin S(21)68 is an essential part of the phage Phi 21 lytic protein system to release the virus progeny at the end of the infection cycle. It is known as the simplest natural timing system for its precise control of hole formation in the inner cytoplasmic membrane. Pinholin S(21)68 is a 68 amino acid integral membrane protein consisting of two trans membrane domains (TMDs) called TMD1 and TMD2. Despite its biological importance, structural and dynamic information of the S(21)68 protein in a membrane environment is not well understood. Systematic site-directed spin labeling and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopic studies of pinholin S(21)68 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) proteoliposomes are used to reveal the structural topology and dynamic properties in a native-like environment. CW-EPR spectral line-shape analysis of the R1 side chain for 39 residue positions of S(21)68 indicates that the TMDs have more restricted mobility when compared to the N- and C-termini. CW-EPR power saturation data indicate that TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S(21)68. A tentative structural topology model of pinholin S(21)68 is also suggested based on EPR spectroscopic data reported in this study.