To investigate the role of Phe101, a component of a base recognition site (B2 site) of a base-nonspecific RNase Rh from Rhizopus niveus, we prepared several enzymes mutated at this position, F101W, F101L, F101I, F101A, F101Q, F101R, and F101K, and their enzymatic activities towards RNA, 16 dinucleoside phosphates, and 2', 3'-cyclic pyrimidine nucleotides were measured. Enzymatic activity toward RNA of F101W, F101L, and F101I were about 7, 20, and 3.8% of the native enzyme, respectively, and those of the other mutants were less than 1% of the RNase Rh. Similar results were also obtained with GpG as substrate. Thus, it was concluded that Phe101 is a very important residue as a component of the B2 site of RNase Rh, and its role could be replaced by Leu, then Trp and Ile, though in less effectively. The results suggested that some kind of interaction between B2 base and the side chain of amino acid residue at the 101th position, such as pi/pi or CH/pi interaction is very important for the enzymatic activity of RNase Rh. The mutation of Phe101 markedly affected the enzymatic activity toward dinucleoside phosphates and polymer substrates, but only moderately the rate of hydrolysis of cyclic nucleotides, indicating the presence of secondary effect of the mutation on B1 site.