To clarify whether there is a bias in the V-D-J combination of T cell receptor (TcR) genes, J beta gene usage has been investigated in a total of 743 TcR beta genes of V beta 2, V beta 8.2, and V beta 14 families expressed in C57BL/6 mouse spleens. Genes of TcR beta chains, amplified by a reverse transcriptase-polymerase chain reaction, were individually cloned into plasmids. Cloned genes (61 to 106), randomly selected in each respective V beta family from three different mice, were tested by means of hybridization with 12 oligo DNA probes which were designed to differentiate 12 murine functional J beta gene segments. The results are enumerated below. (1) The J beta 2.6 gene segment was found to be most frequently used (V beta 2, 19.8%; V beta 8.2, 21.2%; and V beta 14, 19.2%). In contrast, usage of the J beta 1.6 gene segment was most infrequent (V beta 2, 1.9%; V beta 8.2, 2.9%; and V beta 14, 0.5%); (2) High frequency of the J beta 2.1 gene segment and low frequency of the J beta 1.3 and J beta 1.5 gene segments were also observed; (3) The J beta 2 cluster was used in preference to the J beta 1 cluster (usages of the J beta 2 cluster: V beta 2, 67.8%; V beta 8.2, 65.9%; and V beta 14, 70.4%); and (4) These biases were generally common to all three V beta families examined and differences among individual mice were mostly small. Considering these findings, we conclude that the TcR J beta gene segments in C57BL/6 mice splenocytes are selected with a bias, but are selected independently of the V beta families.