Flow cytometry was used to estimate the relative nuclear DNA content of pycniospores from 85 collections of 13 species of rust fungi. For a given sample, 10,240 fluorescent events were measured. Sufficient pycniospores could be obtained from as little as one well-developed pycnial cluster, Numbers of fluorescent events plotted against intensity of fluorescence usually exhibited a single well-defined peak with coefficients of variation of 10% or less. Results confirmed and extended data for small numbers of basidiospores obtained earlier with the microscope photometer. DNA content relative to that of a Puccinia hordei standard was estimated to be as follows (in order of increasing DNA content): P. lagenophorae, 53%; P. graminis, 56%; P. coronata, 64%; P. sorghi, 84%; P. hordei, 101%; P. recondita, 105%; Uromyces hippomarathricola, 107%; U. reichertii, 120%; Tranzschelia pruni-spinosae, 150%; P. allii, 164%; P. helianthi, 185%; U. vignae, 336%; and U. appendiculatus, 346%. Within P. hordei, collections from Hordeum bulbosum had higher DNA content than collections from other telial host species; within P. recondita, collections from each of four telial host species differed, suggesting that these fungi are diverging genetically on some telial host species.