Glomalin, a family of proteins extractable from soil in neutral to basic citrate by autoclaving, has been associated with improved soil aggregate stability, and is purportedly of arbuscular mycorrhizal fungal (AMF) origin. We extracted glomalin from soil aggregates of two size classes (0.25-2 mm, 2-8 mm) from replicated experimental tillage trials in 5 locations in NY State at 4 sampling dates through the 2004 corn growing season. Glomalin concentrations in the operationally- defined easily extractable (EEG) and total (TG) glomalin pools were quantified using a Bradford dye binding assay, with bovine serum albumin (BSA) and two alternative standards. Glomalin concentrations responded differently to tillage and harvest treatments, but the significance and direction of these responses varied by sampling date. Aggregate stability likewise varied with glomalin concentration, but differently across the season. Correlations between glomalin concentrations and soil aggregate stability were notably lower than those reported by others, and also varied by sampling date. We tested the validity of the assumption that glomalin is exclusively of AMF origin with a spiking experiment, while developing a more appropriate standard for Bradford quantification of glomalin concentrations. We added purified proteins, or organic matter of various sources, in realistic amounts, to quartz sand, and subjected these to the established glomalin extraction protocol. Protein yields of these extracts were significantly greater than those of reagent blanks, and in some cases were comparable to protein yields from soils analyzed in this study. These results highlight the need to revisit basic assumptions in discussions of the nature and properties of glomalin.