Two mutants of coxsackievirus B1 (CVB1), CVB1c and CVB1e, with mutations in the stem-loop H of 5'-UTR were generated by site-directed mutagenesis. The A at nt579 of CVB1c was substituted by G. The U at nt573 and A at nt579 of CVB1e were substituted by A and G. The virulences of these mutants had been assessed by means of cytopathic effect (CPE), plaque formation, one-step growth curve, and 50% lethal dose (LD50) assays. The pathogenesis of these mutants was evaluated by attacking suckling Balb/c mice. Plaque assay and one-step growth curve showed that the replication of CVB1c and CVB1e on HeLa cells was significantly faster than that of their prototype CVB1n. Data of CPE assay, LD50, and pathological examination showed that CVB1c and CVB1e were more virulent than CVB1n. These data showed that mutation at nt579 (A-->G) alone and mutations at nt579 (A-->G) and nt573 (U-->A) together within 5'-UTR caused significant augment of the virulence and pathogenesis of coxsackievirus B1, and suggested that nt573 and nt579 might be molecular determinants for the virulence of coxsackievirus B1.