Mouse hepatitis viruses (MHV) are coronaviruses which cause various infections in mice affecting lung, intestine, liver, and other organs as well as the central nervous system. The replication of three different MHV strains was studied in mouse dissociated spinal cord cultures containing differentiated neurons and nonneuronal cells (NN) (including astrocytes). Cell tropism and maturation of each virus strain was analyzed by immunolabeling methods using antisera to the virion or to purified membrane glycoproteins (E1 and E2) and by electron microscopy (EM). Wt-JHM, which causes acute encephalitis in mice, produces acute cytopathic changes in both neurons and NN cells. In neurons, virions mature in smooth ER cisternae closely associated to the Golgi apparatus. As judged by EM, fewer virions are produced by neurons than NN cells and neurons do not fuse or stain for E2 as do NN cells. NN cells contain large inclusions made of nucleocapsid strands. A temperature-sensitive mutant of JHM, Ts8-JHM, which causes demyelination in mice, infects NN cells but not neurons. Infected NN cells synthesize E1 and E2, and contain large inclusions but few mature virions, even at permissive temperatures. These inclusions appear granular and rarely contain nucleocapsid strands in contrast to wt-JHM infection. NN cells infected with this mutant also display numerous membrane whorls. The hepatotropic strain A59 lacks tropism for neurons and primarily infects NN cells, thus resembling ts8-JHM. Infected NN cells become loaded with intracytoplasmic virions which are secreted from the cells. E1 can only be detected in the perinuclear area of these cells while E2 rapidly spreads throughout the cytoplasm. The cytoplasm of A59 infected NN cells frequently contains large tubular structures often in the lumen of the RER. In conclusion, in primary CNS cultures consisting of neurons and NN cells: (1) wt-JHM replicates in both neurons and NN cells but has different effects on these cells; (2) Ts8-JHM exhibits no productive infection of neurons, and in NN cells appears to be defective in assembly and to stimulate membrane synthesis; (3) A59 also shows tropism restricted to NN cells which produce many viruses and display differential distribution of the two virion glycoproteins. Thus, in the absence of the immune system, the MHV strains assayed exhibit differences in viral tropism, cytopathic changes, and viral assembly in CNS cells, and these differences may account for the different disease patterns.