Periphyton assemblage data collected from 233 stream site-visits (49 in 1993, 56 in 1994, and 128 in 1995) throughout the Mid-Appalachian region were used to develop a periphyton index of biotic integrity (PIBI) based on 1) algal genera richness; 2) the relative abundances of diatoms, Cyanobacteria, dominant diatom genus, acidophilic diatoms, eutraphentic diatoms, and motile diatoms; 3) chlorophyll and biomass (ash-free dry mass) standing crops; and 4) alkaline phosphatase activity. Thirty-seven diatom genera and 38 non-diatom genera were collected. The relative richness and relative abundance (RA) of these genera were used to calculate the RA metrics of the PIBI. PIBI scores ranged from 48.0 to 85.1 among the 233 site-visits with an overall regional mean (±1 SE) of 66.1 ± 0.5. The 10 metrics and the PIBI were correlated with 27 chemical, 12 physical habitat, and 3 landscape variables. Overall, PIBI was inversely correlated with stream depth, stream water color, and Fe. Component metrics were significantly correlated with several chemical (Al, acid neutralizing capacity, Cl, Fe, Mn, N, Na, P, pH, Si, SO4, total suspended solids), physical habitat (channel embeddedness, riparian disturbances, stream depth, stream width, substrate composition), and landscape (% of the watershed in forest, agriculture, and urban land uses) variables. Canonical correlation analysis revealed significant correlations between the 10 PIBI metrics and 4 significant environmental gradients related to general human disturbances (stream acidity, stream substrate composition, and stream and riparian habitat). Analysis of variance revealed significant differences in PIBI scores for lowland vs highland streams, and among stream orders. Annual differences were explained by differences in the proportions of sampling sites in lowland streams in each year. The univariate distribution of PIBI scores was used to set threshold PIBI values for the assessment of ecological condition in Mid-Appalachian streams.