In c mu- B220+ Thy-1- FL2-52-2, an immature B cell line transformed with a temperature-sensitive mutant of Abelson murine leukemia virus(ts OS-59), Thy-1 antigen expression was induced after the shift of the culture temperature from a permissive(35 degrees C) to a non-permissive temperature(39 degrees C), and a Thy-1+ subclone, FL2-52-2-1 was isolated by limiting dilution. Furthermore, since a population of Thy-1+FL2-52-2-1 lost Thy-1 antigen expression during culture at a non-permissive temperature, Thy-1- FL2-52-2-1-1 was isolated from the cultured cells. Methylation analysis by Southern blotting experiments showed that 5' region of the Thy-1 gene was methylated in Thy-1- FL2-52-2 but demethylated in Thy-1+ FL2-52-2-1 and Thy-1-FL2-52-2-1-1. To determine whether or not there exist intracellular regulatory factors responsible for Thy-1 gene expression, Thy-1a allele was transfected into Thy-1(Thy-1.2-) FL2-52-2 and Thy-1- FL2-52-2-1-1. The transfected Thy-1a allele was expressed in Thy-1- FL2-52-2, but not in Thy-1- FL2-52-2-1-1, indicating the presence of the intracellular regulatory factors requisite for Thy-1 gene expression in Thy-1- FL2-52-2. It appeared that in Thy-1- FL2-52-2-1-1, Thy-1 gene was not expressed because of the absence of the intracellular regulatory factors although the 5' region of the Thy-1 gene was demethylated. These results indicated the existence of at least two regulatory mechanisms of Thy-1 gene expression in immature B cells: methylation of the 5' region of the Thy-1 gene and intracellular regulatory factors.