Shiverer (shi) is an autosomal recessive mutation in mice that results in hypomyelination in the central nervous system (CNS) but normal myelination in the peripheral nervous system (PNS). Myelin basic proteins (MBPs) are virtually absent in both PNS and CNS. It is not known whether the cellular target in the PNS is the myelin-forming Schwann cell or another cell type which secondarily affects the Schwann cell. To determine the cellular target of the shi gene, we have adapted tissue culture techniques that allow co-culture of pure populations of mouse sensory neurons of one genotype with Schwann cells and fibroblasts of another genotype under conditions that permit myelin formation. These cultures were stained immunocytochemically as whole mounts to determine whether MBPs were expressed under various in vitro conditions. In single-genotype cultures, presence or absence of MBPs was consistent with earlier in vivo results: +/+ cultures were MBP-positive and shi/shi cultures were MBP-negative. In mixed-genotype cultures, visualization of MBPs in myelin accorded with the genotype of the non-neuronal Schwann cells and fibroblasts and not with the neurons--those cultures that contained +/+ non-neuronal cells were MBP-positive and those with shi/shi non-neuronal cells were MBP-negative, independent of the neuronal genotype. These results rule out neurons or circulating substances as mediators of the influence of the shi genetic locus on MBP synthesis and deposition in peripheral myelin.