730 The rejection of xenografts is primarily mediated by anti-donor antibodies. We have previously demonstrated that the early phases of this reaction are controlled by a closely-related family of Ig germline VH genes(VHHAR) expressed by IgM xenoantibodies. We have recently extended these studies to investigate whether the use of the VHHAR gene family is observed during the maturation of the anti-donor antibody response and if this response exhibits a switch from IgM to IgG antibody isotypes. Total RNA was extracted from splenocytes harvested from naive animals and xenograft recipients of hamster hearts at days 4, 8, 21 and 28 post-transplantation. cDNA libraries were prepared by using a RT-PCR technique, followed by PCR amplification with a primer (RVH1) specific for VHHAR genes and a second primer (RCG) specific for the common region in the CH2 of γ1, γ2a, γ2b and γ2c. The cDNA libraries were also used to estimate the precursory frequency of VHHAR gene family using a quantitative PCR technique. A plaque lift hybridization technique was used to establish the ratios of IgG subclasses used by antibodies mediating the response. At 21 days post transplantation, IgG antibodies expressing VHHAR genes exhibited a six-fold increase in usage; the IgG1 isotype of HAR-IgG accounted for the largest increase. Nucleotide sequence analysis demonstrated the existence of all HAR-IgG isotypes in naive rats, and the VH regions of IgG1 genes exhibited sequence identities with HAR genomic germline genes of 92.4 to 97.4% and 93.5 to 98.9% at 21 and 28 days post transplantation. VH germline progenitors of these clone could not be identified in rat liver genomic DNA, suggesting affinity maturation in their recognition of the target antigens due to somatic mutations. However, Ig VDJ gene rearrangements identical to those seen in IgM antibodies capable of causing hyperacute rejection are found in HAR-IgG library. We conclude that early stages of the humoral response to xenografts represents a T cell-independent pathway of antibody production by the graft recipients, while later stages contains both T cell-dependent and independent pathways. Additional studies in progress will address the relative contribution of these pathways to graft rejection in vivo.