Hyaluronic acid (hyaluronan) is a high molecular weight glycosaminoglycan composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine. The molecular weight of hyaluronan is important for its rheological property, biological function, and application. However, genes important for regulation of hyaluronan production or molecular weight remain poorly understood. Here, we address the roles of two predicated hyaluronate lyase-encoding genes, hylB and hylZ in Streptococcus zooepidemicus. Semi-quantitative RT-PCR assays showed that hylZ was constitutively expressed while the expression level of hylB was growth-phase dependent. Using recombinantly expressed 6His-HylB and -HylZ protein, enzyme assays revealed that HylB was a hyaluronate lyase, and its K (m) and V (max) were 0.57 mu M and 1.43 mM min(-1), respectively. 6His-HylZ showed no activity against hyaluronan while it hydrolyzed pNp-beta-GlcNAc suggesting that HylZ was a beta-N-acetylglucosaminidase. Under the optimal conditions (pH 6.0 and 50 A degrees C), the K (m) and V (max) for 6His-HylZ to degrade pNp-beta-GlcNA were 1.16 mM and 26.18 mu mol min(-1) mg(-1), respectively. Characterization of Delta hylB and Delta hylZ demonstrated that loss of hylB or/and hylZ had no significant effects on cell growth, lactic acid, and hyaluronan yields. Significantly, as compared to the wild type, Delta hylB produced hyaluronan with an 18 % increase in molecular weight. Our results strongly suggest that hylB encodes a hyaluronate lyase while hylZ encodes a beta-N-acetylglucosaminidase. hylB-deficient strain has the potential to produce high molecular weight hyaluronan.