A highly neurovirulent murine coronavirus JHMV (wild-type [wt] JHMV) is known to spread from cells infected via the murine coronavirus mouse hepatitis virus receptor (MHVR) to cells without MHVR (MHVR-independent infection), whereas a mutant virus isolated from wt JHMV, srr7, spread only in an MHVR-dependent fashion. These observations were obtained by the overlay of JHMV-infected cells onto receptor-negative cells that are otherwise resistant to wt JHMV infection. MHVR-independent infection is hypothetically thought to be attributed to a naturally occurring fusion activation of the wt JHMV S protein, which did not occur in the case of srr7. Attachment of S protein on cells without MHVR during the S-protein activation process seems to be a key condition. Thus, in the present study, we tried to see whether wt JHMV virions that are attached on MHVR-negative cells are able to infect those cells. In order to make virions attach to the cell surface without MHVR, we have used spinoculation, namely, the centrifugation of cells together with inoculated virus at 3,000 rpm for 2 h. This procedure forces viruses to attach to the cell surface, as revealed by quantitative estimation of attached virions by real-time PCR and also facilitated wt JHMV infection to MHVR-negative cells, but failed to do so for srr7. Virions of both wt and srr7 attached on MHVR-negative cells by spinoculation were facilitated for infection in the presence of a soluble form of MHVR that induces conformational changes of both wt and srr7. It was further revealed that wt JHMV S1, but not srr7, was released from the cell surface when S protein was expressed on cells. These observations support the hypothesis that attachment of the virion to MHVR-negative cells is a critical step and that a unique feature of wt JHMV S1 to be released from S2 in a naturally occurring event is involved in an MHVR-independent infection.