Autoimmune reactions associated with MS involve genetic and environmental factors. Because murine coronaviruses induce an MS-like disease, the human coronaviruses (HCoV) are attractive candidates as environmental factors involved in a demyelinating pathology. We previously reported the isolation of HCoV-229E/myelin basic protein (MBP) cross-reactive T-cell lines (TCL) in MS patients. To investigate antigenic cross-reactivity at the molecular level, 155 long-term T-cell clones (TCC) were derived from 32 MS patients by in vitro selection with MBP, proteolipid protein (PLP) or HCoV (strains 229E and OC43). Overall, 114 TCC were virus-specific, 31 were specific for myelin Ag and 10 other were HCoV/myelin cross-reactive. Twenty-eight virus-specific TCC and 7 myelin-specific TCC were obtained from six healthy donors. RACE RT-PCR amplification of the Vbeta chains of five of ten the cross-reactive TCC confirmed clonality and sequencing identified the CDR3 region associated with cross-reactivity. Our findings have promising implications in the investigation of the role of molecular mimicry between coronaviruses and myelin in MS as a mechanism related to disease initiation or relapses.