The chloroform fumigation-incubation method for estimating soil microbial biomass is applicable to most soils; but, problems are associated with its use with extremely acidic soils and with soils amended with carbonaceous substrates. To evaluate the basis for these difficulties, this project was commenced with the objectives of (a) evaluating the variation in the fungal respiration in fumigated and native acidic forest soil and of (b) assessing the effect of chloroform fumigation on microbial respiration and invertase activity in this soil amended with glucose or corn stover. The soil samples were collected from the O-horizon (pH 3.5) and A-horizon (pH 3.5) of a Lakehurst soil series (a mesic coated Haplaquodic quartzipsamment). Cycloheximide and streptomycin inhibition of glucose respiration indicated that in native O-horizon soil, the fungal to bacterial activity ratio was approximately 82:12. During the incubation period following fumigation, fungal activity ranged from 92 to 99% of total glucose respiration in fumigated O-horizon soil samples and 83 to 90% in the nonfumigated soils. Comparable values for the A-horizon soils were 85 to 90% in the fumigated soils and 33 to 84% in the native samples. Return of glucose metabolism to pre-fumigation rates occurred within 2 days after fumigation in the chloroform treated O-horizon soils whereas 10 days were required in the A-horizon soils. Chloroform fumigation of soils receiving corn stover or glucose caused a greater production of CO2 and more invertase activity due to the amendment than was detected in comparable nonfumigated soil samples. These data suggest that differences in basic microbial population activity ratios between chloroform-fumigated acidic soils and unfumigated control soil samples contribute to the difficulties of applying the chloroform fumigation-incubation method to extremely acidic soils and that the method is likely invalid for use with extremely acidic soils receiving inputs of carbonaceous compounds.