Two strains of minute virus of mice (MVM) show different host-cell specificities. MVM(i) grows in T lymphocytes whereas MVM(p) is fibroblast-specific. By constructing recombinant viral DNAs between the genomes of the two strains, we have shown that two segments of the MVM(i) genome are required for lytic viral growth in T lymphocytic EL4 cells. One segment (iE) was found between nucleotides 1084 and 2070, in a region encoding the early viral proteins and containing mRNA splice signals and the late P39 promoter. The other (iL) was between nucleotides 3523 and 4339 in the region coding for capsid protein. The P39 promoters within the E segment from MVM(i) or MVM(p) were equally active in transfected EL4 cells. However, pE-containing MVM DNA produced more NS2 mRNA than iE-containing DNA, apparently the result of virus-strain-specific differences in the regulation of splicing.