Mouse immunoglobulin epsilon chain gene was cloned from DNA of a hybridoma producing anti-dinitrophenyl IgE, which was constructed by fusing a spleen cell of a BALB/c mouse with a variant clone of MOPC21 myeloma (IgG1 producer). Because a given active heavy chain constant region (CH) gene is linked to a heavy chain joining segment (JH) gene at its 5' side, the expressed C epsilon gene of the hybridoma was cloned from a phage library containing partial Sau3A digests of IgE hybridoma DNA by using a J gene fragment as a probe. Among 6 X 10(5) phages screened, five positive clones were obtained and three of them were identified as C epsilon gene clones by restriction mapping, Southern blot hybridization, R-loop formation, and partial nucleotide sequence determination. The determined nucleotide sequence predicted the amino acid sequence which resembles a part of the CH3 domain of human epsilon chain. The deletion profile of the C epsilon gene in various myelomas expressing different CH genes indicates that the C epsilon gene is located between the C gamma 2a and C alpha genes. The linkage (5'-epsilon-alpha-3') was directly confirmed by molecular cloning of the overlapping chromosomal segments from newborn mouse DNA.