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Ergot caused by Claviceps purpurea results in the contamination of rye grain by sclerotia that contain alkaloids toxic to humans and other mammals. Ergot incidence and severity are affected by various factors including the availability of pollen during flowering. To test the presence of variation for ergot resistance due to anatomical and/or biochemical factors in rye, we studied cytoplasmic-male sterile (CMS) inbred lines of the Petkus gene pool (two sets each having 30 lines) and their testcrosses with maintainer tester lines (Set I crossed with two and Set II with a third tester line). Sixty-four CMS lines (60 lines, three testers in CMS form, one standard CMS line) and 90 CMS testcrosses were evaluated in four and three environments, respectively, for ergot severity measured as sclerotia weight per ear under pollen isolation and artificial spray inoculation. We also analysed the concentration of the six most important alkaloids and their isomeres in 25 lines. A very high ergot severity was achieved, and despite that genotypic variance among the 64 CMS lines was significant ( P < 0.01). In testcrosses, genotypic variance was smaller, even being not significant in testcrosses of one tester. Genotype × environment interaction variances and correlation coefficients between lines and their testcrosses were significant ( r = 0.56–0.75, P < 0.01) in all instances. Most prevalent alkaloids were ergosin, ergocristin, and ergotamin. There were no significant genotypic differences among lines for any alkaloid or isomer, but total alkaloid concentration showed genotypic variation with low significance level ( P < 0.1). In conclusion, we detected genotypic differences for resistance in CMS rye based on variable response of ovaries during infection process that can be exploited by multi-locational evaluation and selection to develop ergot resistant hybrids. [ABSTRACT FROM AUTHOR]