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The aim of this research was to empirically validate constructs for evaluation of teachers' attitudes toward usage of educational software in chemistry teaching. Questionnaire with items transformed from UTAUT (Unified theory of acceptance and use of technology) and other technology acceptance theories were filled in by 556 Czech chemistry teachers. All constructs passed recommended .7 thresholds of Cronbach's alpha so they can be used in acceptability researches before and after introduction of educational software or building models. However, analyses of effect sizes show that there are not only differences between users and all nonusers generally, but also prove differences between various types of nonusers. Nonusers were established as a) those who had used educational software and abandoned it; b) those who do not use educational software, but are planning to use it in the future, and c) those who do not use educational software and have no intentions to use it. An unexpected finding reveals that differences among subgroups of nonusers can be even larger than between users and nonusers, especially the group c) is an outstanding group. Consequently, factors and their influence on the acceptance and use of educational software in chemistry teaching should be explored for each group separately.