BACKGROUND AND OBJECTIVE: Attention-deficit/hyperactivity disorder (ADHD) is the most frequent psychiatric disorder in children and adolescents and is often treated with methylphenidate (MPH), resulting in MPH exposure in more than 1% of all children in many countries. A 2005 report on cytogenetic effects in peripheral lymphocytes from 12 ADHD children treated for 3 months with MPH raised questions about its genetic toxicity and potential carcinogenicity. In 2007, we described no elevated micronucleus frequency in 21 children after 3 months of MPH-treatment; since the difference between the two studies could not be explained we now enlarged the overall sample size, and added a healthy control group, a new chronically treated group and positive control slides. Furthermore, micronuclei were analyzed in a second tissue, buccal mucosa. STUDY PARTICIPANTS: A healthy control group (23 individuals), a chronically MPH-treated (>12 months) group (21 patients), and a drug naïve group of ADHD-affected children (26 patients), which was analyzed again after 3 months (17 patients) and 6 months (11 patients), provided samples for analysis of micronucleated lymphocytes. With inclusion of 14 previously obtained blood samples, an overall group size of 31 patients was reached for the comparison of the 3 months observation time with before for micronucleated lymphocytes. For buccal mucosa cells, an additional inclusion of 10 more chronically treated patients (no lymphocytes donated) yielded sample numbers of 22 (healthy), 17 (chronically treated), 23 (ADHD drug naïve), 14 (3 months) and 11 (6 months). RESULTS: No significant alteration in genomic damage as seen as micronucleus frequency in peripheral lypmphocytes or buccal mucosa cells was detected after MPH treatment. CONCLUSIONS: No indication for genomic damage induced by MPH was obtained in this study, as in our previous study. Together with our previous study, our overall number of MPH-treated patients is now 68 (30 chronically treated, 38 prospectively followed), plus 23 healthy controls. Ongoing studies in the USA, as well as continuation of recently published epidemiological cancer incidence analysis should provide additional reassurance for MPH-treated ADHD patients.