This study evaluates associations between internalized homonegativity and demographic factors, drug use behaviors, sexual risk behaviors, and HIV status among men who have sex with men (MSM) and with men and women (MSM/W). Participants were recruited in Los Angeles County using respondent-driven sampling (RDS) and completed the Internalized Homonegativity Inventory (IHNI) and questionnaires on demographic and behavioral factors. Biological samples were tested for HIV and for recent cocaine, methamphetamine, and heroin use. The 722 MSM and MSM/W participants were predominantly African American (44%) and Hispanic (28%), unemployed (82%), homeless (50%), and HIV positive (48%) who used drugs in the past 6 months (79.5%). Total and Personal Homonegativity, Gay Affirmation, and Morality of Homosexuality IHNI scores were significantly higher for African American men than for other ethnicities, for MSM/W than for MSM, for recent cocaine users than for recent methamphetamine users, and for HIV-seronegative men than for HIV-seropositive men. Linear regression showed the Gay Affirmation scale significantly and inversely correlated with the number of sexual partners when controlling for effects of ethnicity/race and sexual identification, particularly for men who self-identified as straight. Highest IHNI scores were observed in a small group of MSM/W (n∈=∈62) who never tested for HIV. Of these, 26% tested HIV positive. Findings describe ways in which internalized homophobia is a barrier to HIV testing and associated HIV infection and signal distinctions among participants in this sample that can inform targeted HIV prevention efforts aimed at increasing HIV testing. © 2009 The New York Academy of Medicine.