We investigated a range of microbiological community assays performed on scrapes of biofilms formed on artificial diffusing substrates deployed in 8 streams in eastern Scotland, with a view to using them to characterize ecological response to stream water quality. The assays considered were: Multiplex Terminal Restriction Fragment Length Polymorphism or M-TRFLP (a molecular method). Phospholipid Fatty Acid or PLFA analysis (a biochemical method) and MICRORESP (TM) (a physiological method) alongside TDI, diatom species, and chlorophyll a content Four of the streams were classified as of excellent status (3-6 mu g/L Soluble Reactive Phosphorus (SRP)) with respect to soluble P content under the EU Water Framework Directive and four were of borderline good/moderate or moderate status (43-577 mu g/L SRP). At each site, 3 replicates of 3 solute diffusion treatments were deployed in a Latin square design. Solute diffusion treatments were: KCl (as a control solute). N and P (to investigate the effect of nutrient enrichment), or the herbicide isoproturon (as a "high impact" control, which aimed to affect biofilm growth in a way detectable by all assays). Biofilms were sampled after 4 weeks deployment in a low flow period of early summer 2006. The chlorophyll a content of biofilms after 4 weeks was 2.0 +/- 0.29 mg/m(2) (mean +/- se). Dry matter content was 16.0 +/- 13.1 g/m(2). The M-TRFLP was successfully used for generating community profiles of cyanobacteria, algae and bacteria and was much faster than diatom identification. The PFLA and TDI were successful after an increase in the sample size, due to low counts. The MICRORESP (TM) assays were often below or near detection limit. We estimated the per-sample times for the successful assays as follows: M-TRFLP: 20 min, PLFA 40 min, TDI 90 min. Using MANOVA on the first 5 principal co-ordinates, all the assays except MICRORESP (TM) showed significant differences between sites, but none of the assays showed a significant effect of either initial stream trophic status (as classified by the EU Water Framework Directive using chemical standards for soluble P), or of the diffusing solute treatment Multiple Procrustes analysis on the ordination results showed that the diatom and M-TRFLP data sets hold distinct, though as yet unexplored, information about the ecological factors affecting stream biofilms. The diatom data were subjected to principal components analysis, to identify which taxa were more strongly influenced by site variables, trophic status or treatment effects. These were Acnanthes lanceolata, A minutissimma, Nitzchia spp., Coccineis spp. and Navicula spp. Further experimentation and data analysis on a larger number of sites, to identify specific M-TRFLP bands that could be used as indicators linked to specific taxa, are desirable.