A long-standing dilemma for doing soil microbial assays is how best to store soil samples between sampling and analysis. There have been virtually no systematic studies of storage effects on the methods used to characterize soil microbial communities and functions. The objective of this study was to determine the effects of various sample handling and storage methods on a range of microbial methods. The design of the experiment was 3 X 5 factorial with three soils (one Ultisol, and sandy and loamy textured Mollisols) by five commonly used sample pretreatments: i) analysis of fresh soil within 24 h after sampling; ii) air dried 48 h and stored 14 days in a sealed plastic bag followed by rewetting (65% WHC) and incubating at 25°C for 14 days; iii) 28 days at 4°C; iv) 28 days at -20°C; and v) 28 days at -80°C. Each of the soil treatments was analyzed (3 replications) for EL-FAME (ester-linked fatty acid methyl ester) and DNA yields, 7 enzyme activities, soil microbial biomass C (fumigation/incubation), and respiration. Total DNA and total EL-FAME concentrations decreased for all storage methods in two of the three soils (DNA) or all soils (EL-FAME). Microbial biomass and respiration indicated no clear trend due to storage temperature, and both decreased with drying/rewetting. Microbial community composition using EL-FAME patterns showed that all storage methods resulted in decreased microbial diversity (Shannon index) and decreased lipid biomarkers for gram-negative microorganisms. Several extracellular enzyme activities (i.e., β-glucosaminidase, cellulase and urease) were either not altered or negatively affected by drying, while storage temperatures either resulted in higher or lower activities. Arylsulphatase and alkaline phosphatase showed predominatly lower activities after storage. Multivariate discriminant analysis indicated that storage at 4°C or -20°C had moderate effects on microbial biomass, microbial respiration and enzyme activities, while soil drying and storage at -80°C caused more pronounced alterations. We concluded that DNA extraction and EL-FAME analyses should be performed on fresh soil, while the preferable storage method appeared to be -20°C. Overall, soil storage affected our results, which suggested outcomes for ecological or manipulative experiments would be altered according to how soil samples were stored. More specifically, storage method for most microbial measurements were obscured or created differences between soils that would not have occurred if fresh soils had been used. Exceptions included microbial biomass C, microbial respiration, arylsulfatase activity and most EL-FAMEs, which typically were altered by storage but differences or rankings between soils remained the same.