Using a known human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase inhibitor (NNRTI), 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole (TBZ NSC 625487) as the lead structure for drug design, a series of novel 1H,3H-thiazolo[3,4-a]benzimidazole derivatives substituted on the benzene-fused ring was prepared. Their in vitro anti-HIV-1 activity, as well as their inhibitory effects on the viral reverse transcriptase, were evaluated. The structure-activity relationships for these compounds are described and the results suggest that the apolar binding pocket of RT, into which the NNRTIs must fit, can accommodate only groups with a limited size and shape.