Laboratory assays for the detection of recent HIV infection for HIV incidence surveillance are essential to HIV prevention efforts worldwide because they can identify populations with a high incidence and allow targeting of resources and monitoring of incidence trends over time. This study describes the development of a novel rapid HIV-1 incidence-prevalence (I-P) test that can be used for the simultaneous detection and discrimination of prevalent (long-term) or incident (recent) HIV infections using a single device. A lateral flow assay was developed that uses a multisubtype recombinant gp41 protein applied at two concentrations of antigen (high and low). Prevalent and incident HIV-1 infections can be distinguished based on differential antibody binding at the two antigen concentrations. High level/high avidity antibodies present in prevalent infections bind to and are detected at both antigen concentrations while low level/low avidity antibodies present in recent HIV infections are detected only at the higher antigen concentration line. A total of 205 HIV-positive specimens with known status (recent=105, long-term=100), including 57 specimens from seroconversion panels, were tested by the rapid I-P assay and the results were compared to the HIV-1 BED capture enzyme immunoassay (CEIA). There was a 95.1% agreement of final classification (recent or long-term) with the BED assay (kappa=0.910) (mean recency period=162 days) and a high correlation between the intensity score of the low antigen line with the BED OD-n (Pearson correlation=0.89). The new rapid I-P test has great potential to simplify HIV surveillance efforts by simultaneously providing information on both HIV prevalence and incidence using a single, rapid test device.