Diagnostic kits for the detection of human immunodeficiency virus (HIV) antibodies have reached an unprecedented number. But choice of an ideal, cost-effective, and rapid test for HIV infection is of immense value for use in developing countries like India, where resources are limited. In this study we have evaluated the performance characteristics of the rapid immunochromatographic HIV test kit First Response HIV 1-2.O. First, the laboratory archived 450 characterized plasma/serum specimens, which were tested on First Response HIV 1-2.O. Second, a total of 134 consecutive voluntary counseling and testing (VCT) specimens were also tested and positive specimens were further confirmed with HIV TRI-DOT. All these VCT specimens were cross-checked with HIV double-enzyme-linked immunosorbent assay (ELISA) (Murex and Vironostika), and the results were matched. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and efficiency of First Response HIV 1-2.O with the 450 characterized specimens was 100% for HIV-1 with reference assay. The results in the VCT algorithm were correlating with double-ELISA. In the HIV-2 analysis, five HIV-2-positive specimens in First Response HIV 1-2.O were found to be HIV-2-indeterminate on Western blot. HIV TRI-DOT was unable to pick up two HIV-2 Western blot-positive specimens. First Response HIV 1-2.O has several advantages: low-cost (U.S. $0.70); only 10 microL of specimen; involves only two steps; room temperature storage; ability to differentiate HIV-1 and 2; and use of whole blood specimen. Hence this test kit could be suitable for initial screening in the HIV testing algorithm in resource-limited settings. J. Clin. Lab. Anal. 22:178-185, 2008. (c) 2008 Wiley-Liss, Inc.