According to the EU Water Framework Directive, benthic macroinvertebrates are considered a biological quality element required for typology and classification of the ecological status of freshwaters. The effects of habitat, season, and sampling effort on three common quality metrics were studied in Estonian streams. More than 900 macroinvertebrate samples from minimally disturbed sites, collected in 1985-2002, were analysed. All metrics were significantly influenced by stream size, bottom substrate, flow velocity, and geographical location. The biological quality was the best 30-50 km from the stream source. The geographical distribution of the values of ASPT (mean sensitivity of a taxon) and DSFI (level of organic pollution) was markedly different from the geographical distribution of the values of NTAXA (taxa richness). All metrics also revealed lower quality for sandy than for stony bottom, and for low than for high flow velocity (except NTAXA). Unlike the other two metrics, ASPT did not depend on sampling season and depended much less on sampling method. Hence it could be recommended as a tool for transforming the biological quality derived from historical non-standard samples into modern terms.