We used immunoglobulin gene transfection to study the effect that substituting an homologous light (L) chain for a parental L chain has on antigen fine specificity and affinity. High-affinity monoclonal anti-digoxin antibodies 26-10 and 40-100 were selected for study because their L chains are 92% homologous (although the H chains differ), and their binding with digoxin and digoxin analogs show very different properties. In order to generate a recombinant transfectoma, the genes encoding the 26-10 H and L chains were cloned. After the sequenced clones had been shown to contain the V gene and the transcriptional control elements, the H and L chain V region genes were subcloned into different expression vectors. Both constructs were transfected into myeloma J558L, a lambda 1 chain producer, to verify that the genetic constructs expressed correctly. The recombined 26-10 antibody was identical to parental 26-10 antibody in fine specificity and affinity. The 26-10 L chain construct was then transfected into a cell line, CR-101, that expresses the 40-100 H chain and a lambda 1 chain. The transfectoma 1E6, secreting 40-100 H chain and 26-10 L chain, was selected. Appropriate gene expression in 1E6 was proven by polymerase chain reaction cloning and sequencing. The fine specificity properties of the 1E6 recombinant derive from both the 40-100 and 26-10 antibodies; however, the affinity of 1E6 is 130 times less than that of the parental antibodies. We conclude that, in 1E6, the H and L chains are codominant in their influence on antigen specificity and that homologous pairing of H and L chains is required for optimal affinity.