Artificial substrata are increasingly used to study periphyton, but their ability to reproduce natural substrata remains controversial. Although many studies have made contemporaneous comparisons of periphyton assemblages on artificial and natural substrata at one or a few sites, no broadly based comparison exists. We therefore surveyed the literature to establish conditions under which artificial substrata satisfactorily mimic both the quantity and the quality of natural periphyton assemblages. In general, epilithon was underestimated by the artificial substrata; epiphyton was overestimated, but less severely. These trends were significantly affected by the time available for colonization of the artificial substrata before sampling, site trophy, ambient temperature, and whether the study was conducted in a lake or in running water. Neither the composition of the substratum nor its orientation appeared important. Natural diatom assemblages were usually well simulated by those on artificial substrata, whereas both epilithic and epiphytic green and blue-green algae were severely underrepresented on the artificial substrata. Since artificial substrata often misrepresent both the quantity and the quality of natural periphyton, they should be used with more caution, especially in intersite and interseason surveys.