To study Hg toxicity on soil microbes and their activities, it is necessary to understand its various forms in soils. The objectives of this study were to investigate Hg speciation in four soil types spiked with Hg (300 mg kg(-1) soil) and its effects on soil microbial respiration and enzymes (amidohydrolases and phosphatase) activities. An assessment of the chemical forms, amounts, reactions, and mobility of Hg in soils and sediments is of significant importance to improve and maintain soil and environmental health and sustainability. Mercury speciation analysis was investigated under acidic and alkaline conditions using a modified sequential procedure, which differentiates Hg into the four distinct fractions. Soil microbial respiration and enzymes activities were determined under laboratory settings, by incubating the soils at 25°C for 30 days, and then determining the amount of CO(2) evolved. Speciation results revealed that the water soluble form was the least, with < 1% of the total Hg in the soil types investigated irrespective of the pH condition, while the residual fraction was the most abundant (> 80%) in Canisteo, Houston, and Ketona soils under acidic conditions and < 35% in Decatur soil. Under alkaline conditions, the residual fraction was ≤ 70% in Canisteo, Houston, and ketona and ≤ 29% in Decatur soil. The exchangeable fraction was the second most abundant fraction in the soils used ranging from 3.7-50.0% under acid conditions and 16.9-52.1% under alkaline conditions indicating that Hg desorption was found to be more favorable under alkaline than acidic conditions. Soil respiration was suppressed by Hg especially at the 100 mg kg(-1) concentration level. Amidohydrolases and phosphatases' response in the presence of Hg was variable. Amidohydrolases were more sensitive to Hg (18-90%) than phosphatase (0-35%) in all soils. This study demonstrated that the forms in which Hg exist in soils may determine its bioavailability and toxicity. Also microbial respiration and enzyme activities are potential bioindicators of heavy metal contamination of the environment.