In many cases, virus-encoded acylproteins appear to localize to specific cellular and viral membranes and to be directly involved with the processes of virus morphogenesis and/or egress from the infected cell. It was therefore of interest to determine whether the major vaccinia virus (VV) myristylprotein, L1R, is specifically associated with one or more of the membranes enveloping various infectious forms of VV virions. To this end, single-membraned intracellular virions (INV) and extracellular enveloped virions (EEV), which are surrounded by at least two distinct membranes, were purified from VV-infected cell lysates. The location of the VV L1R protein was determined by using a monospecific anti-L1R serum to detect the L1R protein by immunoblot in INV- and EEV-containing fractions, by examining the proteinase K sensitivity of the L1R protein in intact INV and EEV particles, and by immunoelectron microscopy. The data obtained clearly indicate that although the L1R protein is a constituent of both the INV and EEV particles, it is exclusively found in the inner INV-specific membrane. These results are discussed with regard to the potential role of the VV L1R protein in the primary intracellular envelopment of infectious VV particles.