Four different-strains of human polyomavirus JC virus (JCV), CY, Taiwan-1, Taiwan-2, and Taiwan-3, have been found in pregnant women and autoimmune disease patients in Taiwan. In this study, we report the cloning and sequencing of the Taiwan-3 JCV, virus isolated from the urine of an immunosuppressed patient with rheumatoid arthritis. The viral genome was amplified by polymerase chain reaction and then cloned into a prokaryotic replicative plasmid, pGEM-7Zf(-). The genomic DNA was sequenced and found to comprise 5,111 base pairs. The enhancer-promoter region of the viral genome lacks a copy of pentanucleotide-A (GGGAA) and pentanucleotide-B (AAAGC) compared to the CY archetypal JCV. There are 108 nucleotides altered in the total genome, excluding the variable part of the enhancer-promoter region, between Mad-1 (the prototype JC virus) and Taiwan-3. The enhancer-promoter region has approximately 25% of the altered nucleotides, resulting in amino acid changes in the open reading frames for I.T. capsid proteins (VP1, VP2, and VP3), and the agno protein. The cloned Taiwan-3, genome will provide an source for physiologic and pathologic investigation of the JCV virus in the future.