BACKGROUND AND OBJECTIVES. Many previous studies showed clear gender differences in the percentages of adults reporting toothbrushing more than once a day. This study evaluated the factors determining gender differences in toothbrushing among Lithuanian middle-aged university employees. MATERIAL AND METHODS. A questionnaire survey was anonymously conducted among 35- to 44-year-old employees (n=862) of four universities in Lithuania in 2005. The response rate was 64% (n=553). Data covered toothbrushing frequency, habitual dental attendance, dental health attitudes and knowledge, and subject's background information. RESULTS. Of all respondents, 68% reported brushing their teeth more than once a day (73% of women and 49% of men, P<0.001) and 51% indicated checkup-based habitual dental attendance (54% of women and 41% of men, P=0.012). The majority reported good dental status as being important to them and poor oral health as injurious to general health. Of all respondents, 44% indicated that "Lack of time is the main reason for incomplete oral self-care" (61% of men and 40% of women, P<0.001). Logistic regression models showed that the strongest factor affecting toothbrushing frequency was the importance of good dental health to them (OR, 1.6; 95% CI, 1.1-2.4; P=0.02) among women and statement that "Poor oral health can be injurious to general health" (OR, 2.6; 95% CI, 1.2-5.5; P=0.01) and checkup-based habitual dental attendance (OR, 2.4; 95% CI, 1.0-5.9; P=0.06) among men. CONCLUSIONS. Due to different determinants affecting toothbrushing frequency among men and women, different oral health motivation programs by gender should be developed.