Herbicides applied to soils potentially affect soil microbial activity. The quantity and frequency of Roundup Ultra [RU; N-(phosphonomethyl)glycine; Monsanto, St. Louis, MO] applications have escalated with the advent of Roundup-tolerant crops. The objective of this study was to determine the effect of Roundup Ultra on soil microbial biomass and activity across a range of soils varying in fertility. The isoproplyamine salt of glyphosate was applied in the form of RU at a rate of 234 mg active ingredient kg(-1) soil based on an assumed 2-mm glyphosate-soil interaction depth. Roundup Ultra significantly stimulated soil microbial activity as measured by C and N mineralization, as well as soil microbial biomass. Cumulative C mineralization as well as mineralization rate increased above background levels for all soils tested with addition of RU. There were strong linear relationships between C and N mineralized, as well as between soil microbial C and N (r2 = 0.96 and 0.95, respectively). The slopes of the relationships with RU addition approximated three. Since the isopropylamine salt of glyphosate has a C to N ratio of 3:1, the data strongly suggest that RU was the direct cause of the enhanced microbial activity. An increase in the C mineralization rate occurred the first day following RU addition and continued for 14 d. Roundup Ultra appeared to be rapidly degraded by soil microbes regardless of soil type or organic matter content, even at high application rates, without adversely affecting microbial activity.