We developed and evaluated an immunoassay for the detection and quantification of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 using electrochemiluminescence technology. The assay had a dynamic range of 50 to 20,000 pg/ml and a lower detection limit equivalent to approximately 10^6.5 HIV-1 RNA copies/ml in culture supernatant. In vitro kinetic replication studies showed that the amount of p7 correlated strongly with the amount of p24 (R² = 0.869; P < 0.0001) and viral RNA (R² = 0.858; P = 0.0009). On the basis of the p7 and RNA concentrations, we calculated the median p7:RNA ratio to be approximately 1,400 p7 molecules per RNA molecule. HIV-1 p7 could be detected and quantified in culture supernatants of both group M subtype A to E viruses and group O viruses. The presence of p7 in vivo was evaluated in 81 serum samples collected from 62 HIV-1-infected individuals. Five samples were p7 positive, whereas 45 samples were HIV-1 p24 positive. Four of the five p7-positive samples were p24 positive as well. p7 could be detected only when serum HIV-1 RNA levels were greater than 10⁶ copies/ml. Anti-p7 antibodies were found in six samples, and all six were p7 negative. In contrast to the in vitro results, it appeared that HIV-1 p7 could not be used as a marker for viral quantification in vivo, since more than 90% of the serum samples were p7 negative. In combination with the low prevalence of anti-p7 antibodies, this may, in turn, be advantageous: the p7 assay may be a good alternative to the p24 assay as the readout system for determination of neutralizing activity against HIV-1 in serum or other fluids containing anti-p24 antibodies.