Properties of the membrane protein (M) of the paramyxovirus simian virus 5 (SV5) isolated from purified SV5 virions, in SV5-infected cells or when expressed from cDNA using a eukaryotic vector (SV40-M) were examined. Kinetic (pulse-chase radiolabeling) studies showed that M protein expressed in SV5-infected and SV40-M recombinant virus-infected cells underwent maturation, detectable as time-dependent acquisition of reactivity with anti-M protein monoclonal antibodies. Kinetic studies using radiolabeled phosphate and studies with the alkylating agent N-ethylmaleimide indicated that the antigenic maturation of the M protein was not due to phosphorylation or disulfide bond formation, respectively. Immunofluorescent antibody staining studies showed a significant difference in staining patterns between SV40-M recombinant virus-infected cells and SV5-infected cells. SV40-M recombinant virus-infected cells exhibited an intensely staining cytoplasmic fibrillar network, whereas in SV5-infected cells, villar and some small granular structures were the only strongly staining structures.