The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.