Most studies on human immunodeficiency virus type 1 (HIV-1) replication kinetics or fitness must rely on a particular assay to initially standardize inocula from virus stocks. The most accurate measure of infectious HIV-1 titers involves a limiting dilution-infection assay and a calculation of the dose required for 50% infectivity of susceptible cells in tissue culture (TCID(50)). Surrogate assays are now commonly used to measure the amount of p24 capsid, the endogenous reverse transcriptase (RT) activity, or the amount of viral genomic RNA in virus particles. However, a direct comparison of these surrogate assays and actual infectious HIV-1 titers from TCID(50) assays has not been performed with even the most conserved laboratory strains, let alone the highly divergent primary HIV-1 isolates of different subtypes. This study indicates that endogenous RT activity, not p24 content or viral RNA load, is the best surrogate measure of infectious HIV-1 titer in both cell-free supernatants and viruses purified on sucrose cushions. Sequence variation between HIV-1 subtypes did not appear to affect the function or activity of the RT enzyme in this endogenous assay but did affect the detection of p24 capsid by both enzyme immunoassays and Western blots. Clear groupings of non-syncytium-inducing (NSI), CCR5-tropic (R5), and SI/CXCR4-tropic (X4) HIV-1 isolates were observed when we compared the slopes derived from correlations of RT activity with infectious titers. Finally, the replication efficiency or fitness of both the NSI/R5 and SI/X4 HIV-1 isolates was not linked to the titers of the virus stocks.