Aims: To find the new microbial parameters explaining the soil fertility from the microbial community viewpoint. Methods and Results: Fatty acid methyl ester (FAME) analysis and terminal-restriction fragment length polymorphism (T-RFLP) analysis were carried out using 16 differently treated plots from the same field that had been kept under different fertilizer management systems since 1984. It was found that organic fertilizer application had small impact, whereas chemical fertilizer application, especially ammonium-nitrogen fertilizer, had strong impact on microbial community structures. Principal component analysis was conducted based on soil chemical and physical parameters, crop yields, FAMEs and terminal-restriction fragments (T-RFs) to provide 10 FAMEs and 10 T-RFs showing strong relation with soil fertility. Conclusion: We defined these 10 FAMEs and 10 T-RFs as 'keystone' biological parameters explaining soil fertility in the soil. Significance and Impact of the Study: This is the first report on the FAMEs and T-RFs related to soil fertility. Both analyses are rapid, inexpensive and reproducible means. As field assessment needs precise and rapid analysis, FAME and T-RFLP analyses and these new parameters are very useful to analyse soil fertility at biological viewpoint.