The entire nucleotide sequence of a hepatitis C virus (HCV) genome (NZL1) of genotype V/3a was determined from overlapping cDNA clones obtained from a human carrier in New Zealand. It comprised 9425 nucleotides (nt) including a 5′-untranslated region of 339 nt, a single large open reading frame encoding a polyprotein of 3021 amino acids, a 3′-untranslated region of 23 nt, and 3′-terminal poly(U) stretches of variable lengths. The NZL1 genome was compared with 15 HCV isolates of other genotypes for which the full-length sequence has been determined. It differed from them by 31.1 to 34.3% in nucleotide sequence identity and by 24.5 to 29.1% in amino acid sequence identity, confirming the distinction of genotype V/3a from the other isolates.