Numerous phenotypes with serologically weak expression of A or B antigen have been found on the surface of red blood cells. Some of these alleles have mutation(s) in the ABO gene coding sequence; most of the mutations are single-nucleotide substitutions leading to an amino acid change. However, these mutated ABO alleles leading to attenuated antigen expression are not well defined. In this study, the cDNAs for the A(1v) and B(1) alleles were first obtained from healthy volunteers and were used as the templates for site-directed mutagenesis to generate expression constructs corresponding to various ABO subtypes including A(1), A(2) (539G>C), A(3) (838C>T), A(1) (829G>A), A(1v) (829G>A), and B(el) (502C>T). K562 erythroleukemia cells were used as the cell study model and were transfected with individual ABO expression constructs. Flow cytometry analysis was then performed to determine the levels of surface antigen expression. Using the relative percentage of antigen-expressing cells as an index for comparison, we found that the levels of A antigen expression for A(1v), A(2) (539G>C), A(3) (838C>T), A(1) (829G>A), and A(1v) (829G>A) were 65.71%, 29.78%, 45.04%, 45.2% and 33.9% of A(1), respectively. Similarly, B antigen expression for B(el) was 90.71% of B(1). The mean fluorescence index (MFI) - defined as the product of percent antigen-expressing cells and the mean cell fluorescence intensity of the population with antigen expression - was also used as an index for comparing the antigen expression level. All ABO subtypes we examined herein displayed a decrease in MFI when compared to A(1) or B(1), respectively. These data thus demonstrate that the point mutation in the coding region of ABO subtypes is fundamental to the weak antigen expression, adding to the complexity of the regulatory mechanisms of ABO gene expression.