The DNA sequence of the mouse H chain V regions from five hybridomas directed against the human tumor Ag tumor-associated glycoprotein-72 (TAG-72) have been determined. This includes a previously determined VH gene sequence from a first-generation anti-TAG-72 mAb, B72.3, and the VH gene sequences from four second-generation anti-TAG-72 mAb, CC49, CC83, CC46, and CC92. A sequence comparison revealed a high degree of shared sequence identity between the five productively rearranged VH genes, suggesting derivation from a common germ line V region gene. In the process of cloning the unrearranged germ line gene, two highly related VH germ line genes were identified and designated VH alpha TAG-1 and VH alpha TAG-2. A comparison of the productively rearranged anti-TAG-72 VH sequences with the two germ line VH genes demonstrated that they were all derived from VH alpha TAG-1. In contrast, the L chain V regions are all derived from separate germ line V region genes. The preferential use of VH alpha TAG-1 in these five mouse hybridomas suggests that VH alpha TAG-1 is a preferred anti-TAG-72 H V chain region germ line gene and that the H chain plays a predominant role in the recognition of this Ag.