A method of inoculating seedlings with the fungus Ramularia collo-cygni, the causal agent of ramularia leaf spot (RLS), an increasingly important problem in barley in Europe and elsewhere, is described. Symptoms of RLS similar to those found in the field were reproduced on seedlings and the fungus was re-isolated from them, fulfilling the third and fourth of Koch's postulates. The method was similar to one used for the related fungus, Mycosphaerella graminicola (anamorph Septoria tritici), a pathogen of wheat. Briefly, plants were sprayed with a suspension of R. collo-cygni mycelium fragments, incubated at 15°C, first in darkness for 48 h then in a 16-h-light/8-h-dark cycle. Disease levels reached saturation when plants were sprayed to runoff with a suspension of 480 cm2 of mycelium, scraped from the entire surface of 7·5 Petri dishes (9 cm diameter) and sieved, in 50 mL water. Growth of seedlings in high light intensity (900 μmol m−2 s−1, 16-h daylength) before inoculation increased disease symptoms, but reduced disease when applied after inoculation. In contrast to M. graminicola, near-ultraviolet light after inoculation reduced symptom development. It is proposed that for the full development of RLS, plants should be grown in a stressful environment before inoculation. Nine barley lines were assessed for their resistance to RLS as seedlings and a subset were tested in field trials with natural infection by R. collo-cygni. There was cultivar-by-isolate interaction in the amount of RLS symptoms on seedlings. RLS levels on adult plants in field plots were correlated with RLS scores on seedlings formed by one isolate but not the other.