The mechanism by which striated muscle internalizes plasmid DNA is unknown. A survey of non-nuclear membrane-associated DNA binding proteins from both skeletal and cardiac muscle revealed several sarcoplasmic reticulum restricted DNA binding species. 32P-DNA overlay and DNA-cellulose chromatography were used to identify membrane-associated DNA binding proteins that may mediate the uptake and expression of plasmid DNA by striated muscle. A total membrane vesicle fraction prepared from rabbit skeletal muscle contained 95-, 60-, and 28-kDa proteins that bound double-strand DNA specifically and with high affinity. The DNA binding proteins appear to originate from the sarcoplasmic reticulum because of their absence in purified sarcolemma vesicles and codistribution with several sarcoplasmic reticulum markers after subcellular fractionation. Several distinguishing biochemical features as well as cross-reactivity with triadin-specific antibodies indicated that the 95- and 60-kDa DNA binding proteins are triadin or proteolytic fragments of triadin, respectively. The role of these sarcoplasmic reticulum proteins in the transport of plasmid DNA is discussed.