Leaf peroxidases of 60 Nicotiana species, 19 cultivars, autopolyploids, interspecific hybrids, and amphidiploids have been compared by polyacrylamide gel block electrophoresis. At least 19 peroxidase bands, four cathodic and 15 anodic, were detected in the species which varied from two bands in N. arentsii to 12 bands in N. tabacum. Tihe cultivars of the latter species failed to reveal any intraspecific variation. Specific difference and varietal resemblance in root peroxidase bands were also observed in nine species and 20 varieties analyzed. Zymograms from autopolyploids and amphidiploids appeared to be identical to that of diploid parents, suggesting that peroxidase banding patterns are independent of ploidy levels. An additive manner of parental peroxidase bands without hybrid enzyme formation in interspecific hybrids and the failure of dissociating peroxidases into subunits lead to a hypothesis that peroxidases in Nicotiana may be controlled by multiple, dominant genes and/or codominant alleles in chromosomes of different genomes. This is in keeping with the lack of relationship between ploidy level and peroxidase banding pattern. Also, species with different chromosome numbers shared many peroxidases in common that possibly reflects a residual homology of peroxidase loci among Nicotiana species. Some species classified in different sections or subgenera but having a common geographic center of origin, showed close similarities in peroxidase zymogram. Results suggest that these species may be closely related in phylogeny, and/or geographic isolation changes the peroxidase genes through mutation and selection. Based on leaf peroxidase zymograms of F 1 hybrids, a putative ancestor of N. tomentosiformis was the progenitor of N. tabacum at its inception.