Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is an RNA molecule that is approximately 9.6 kb in length and encodes a polyprotein that is cleaved proteolytically to generate at least 10 mature viral proteins. Recently, a new HCV protein named F has been described, which is synthesized as a result of a ribosomal frameshift. Little is known about the biological properties of this protein, but the possibility that the F protein may participate in HCV morphology or replication has been raised. In this work, the presence of functional constraints in the F protein was investigated. It was found that the rate of amino acid substitutions along the F protein was significantly higher than the rate of synonymous substitutions, and comparisons involving genes that represented independent phylogenetic lineages yielded very different divergence/conservation patterns. The distribution of stop codons in the F protein across all HCV genotypes was also investigated; genotypes 2 and 3 were found to have more stop codons than genotype 1. The results of this work suggest strongly that the pattern of divergence in the F protein is not affected by functional constraints.