New methods for estimating the numbers of synonymous and nonsynonymous substitutions per site were developed. The methods are unweighted pathway methods based on Kimura's two-parameter model. Computer simulations were conducted to evaluate the accuracies of the new methods, Nei and Gojobori's (NG) method, Miyata and Yasunaga's (MY) method, Li, Wu, and Luo's (LWL) method, and Pamilo, Bianchi, and Li's (PBL) method. The following results were obtained: (1) The NG, MY, and LWL methods give overestimates of the number of synonymous substitutions and underestimates of the number of nonsynonymous substitutions. The major cause for the biased estimation is that these three methods underestimate the number of synonymous sites and overestimate the number of nonsynonymous sites. (2) The PBL method gives better estimates of the numbers of synonymous and nonsynonymous substitutions than those obtained by the NG, MY, and LWL methods. (3) The new methods also give better estimates of the numbers of synonymous and nonsynonymous substitutions than those obtained by the NG, MY, and LWL methods. In addition, estimates of the numbers of synonymous and nonsynonymous sites obtained by the new methods are reasonably accurate. (4) In some cases, the new methods and the PBL method give biased estimates of substitution numbers. However, from the number of nucleotide substitutions at the third position of codons, we can examine whether estimates obtained by the new methods are good or not, whereas we cannot make an examination of estimates obtained by the PBL method. (5) When there are strong transition/transversion and nucleotide-frequency biases like mitochondrial genes, all of the above methods give biased estimates of substitution numbers. In such cases, Kondo et al.'s method is recommended to be used for estimating the number of synonymous substitutions, although their method cannot estimate the number of nonsynonymous substitutions and is time-consuming. These results, particularly result (1), call for reexaminations of some genes. This is because evolutionary pictures of genes have often been discussed on the basis of results obtained by the NG, MY, and LWL methods, which are favorable for the neutral theory of molecular evolution.