A brief, 8-item assessment of HIV treatment knowledge and action was developed and evaluated. Patients with HIV were interviewed in 2001 at infectious disease clinics in Shreveport, LA and Chicago, IL. Analyses on demographics, self-reported medication adherence, and responses to the Rapid Estimate of Adult Literacy in Medicine (REALM) and new, Brief Estimate of Health Knowledge and Action-HIV version (BEHKA-HIV) were performed in 2007. The BEHKA-HIV demonstrated high internal consistency, and construct validity. Lower scores on the BEHKA-HIV were independently associated with poorer rates of HIV medication adherence, scores 4-5 out of 8, AOR 2.6 (95% CI 1.9-3.6), and scores 0-3, AOR 11.4 (95% CI 8.2-15.9), as were the lowest scores on the REALM, AOR 3.3 (95% CI 1.3-8.7). The BEHKA-HIV is a psychometrically sound tool for assessing health knowledge and action regarding HIV treatment, and predicting non-adherence to HIV medications.