To further study the molecular characteristics of group (gp) C rotaviruses, we produced, cloned, and sequenced cDNA to gene 5 of the Shintoku strain of bovine gp C rotavirus. The resulting clone was specific for gene 5 and was genetically related to the human and porcine gp C rotaviruses, as demonstrated by Northern blot hybridization analysis. The Shintoku gene 5 is 1352 nucleotides in length and has one open reading frame encoding a polypeptide of 395 amino acids with a predicted molecular mass of 44.5 kDa. Comparative sequence analysis indicated that: (i) the Shintoku gene 5 protein shared 88.4 to 90.6% homology with the VP6 of the human (Bristol and 88-220) and porcine (Cowden) strains of gp C rotaviruses, but only low homology with the VP6 of bovine gp A (RF) and human gp B (ADRV) rotaviruses (41.3 and 16.3%, respectively); (ii) the predicted secondary structure was highly conserved among the gene 5 proteins of the bovine, porcine, and human gp C rotaviruses; and (iii) seven highly conserved regions were identified for the first time in the deduced primary amino acid sequences of gene 5 of gp C and gene 6 of gp A rotaviruses. However, only three of these highly conserved areas were present in the regions of VP6, where the secondary structure was predicted to be similar for the rotavirus strains examined. These three regions may contribute to common epitopes between the two groups of rotaviruses. Our results, in comparison with data for other rotaviruses, indicate that gene 5 of the bovine gp C rotavirus codes for the major inner capsid protein (VP6).