Indeterminate pattern of results in Western blot (WBI) for human immunodeficiency virus type-1 (HIV-1) infection may represent early HIV-1 infection or may be non-specific in origin. This issue can be resolved by follow up testing upto at least 6 months resulting in psychological distress as well as in high drop out rates among those undergoing investigation pointing out the need for additional parameters that could help in determining the status of HIV-1 infection at the time of initial testing itself in individuals with WBI pattern. The objectives of the present study were: (i) to determine the frequency of HIV-1 infected individuals in a group of professional donors showing WBI patterns in initial testing on the basis of follow up serological studies; (ii) to find out if any HIV related epidemiological or serological characteristics recorded at the time of initial testing could be considered as predictor for HIV-1 infection in WBI specimens; and (iii) to evaluate two alternative serodiagnostic strategies for HIV-1 infection viz. multiple EIAs based on different antigen preparations/principles and a line immunoassay (LIA) employing recombinant antigens in resolving status of HIV-1 infection in specimens showing WBI results at initial testing. Professional donors with WBI patterns belonging to EIA reactive and EIA nonreactive groups were subjected to study of epidemiological profile, prevalence of sexually transmitted diseases (STD) markers and follow up serological testing for HIV-1 at 6, 12, 24 and 48 weeks intervals to record any seroconversion. The initial and follow up specimens from the donors with initial WBI results were subjected to two EIAs (one based on dot immunoassay using synthetic HIV-1 antigens and other based on microwell EIA using recombinant HIV-1 proteins) as well as to LIA. Professional donors with initial WBI results, from the EIA reactive group had higher proportion of unmarried individuals (90.3%), with history of heterosexual promiscuity (75%) and visit to STD clinics (36.1%) compared with the WBI donors from the EIA nonreactive group (72.7, 42.4 and 12.1%, respectively, P values < 0.001). Prevalence of antitreponemal antibodies was higher in the former group (16.7%) compared with the later group (1.5%, P value < 0.002). Seroconversion was recorded in 4 (7.3%) out of 55 EIA reactive WBI donors from the EIA reactive group that were characterised by high optical density (OD) values in EIA, 'p24 only' pattern of band in WB and positivity by LIA at the time of initial testing. LIA was found to be more reliable test compared with combination of EIAs to determine status of HIV-1 infection in WBI specimens at the time of initial testing. The present study points out that parameters like history of heterosexual promiscuity, prevalence of STD markers, high OD values in screening EIA, 'p24' only pattern of bands in WB and positivity by LIA could have individual predictive values for HIV-1 infection in specimens showing WBI pattern of results at initial testing.