The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4 to 72.5%. Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region. These genes were assembled by PCR from synthetic oligonucleotides and expressed in Escherichia coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n = 91) obtained from patients infected with HCV genotypes 1 through 6. All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive serum samples. Although a variable degree of genotype-specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies. Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions. This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.