Diagnosis of COVID-19 by PCR offers high sensitivity, but the utility of detecting samples with high cycle threshold (CT ) values remains controversial. Currently available rapid diagnostic tests (RDTs) for SARS-CoV-2 nucleocapsid antigens (Ag) have sensitivity well below PCR. The correlation of Ag and RNA quantities in clinical nasopharyngeal (NP) samples is unknown. An ultrasensitive, quantitative electrochemiluminescence immunoassay for SARS-CoV-2 nucleocapsid (the MSD S-PLEX SARS-CoV-2 N assay) was used to measure Ag in clinical NP samples from adults and children previously tested by PCR. The S-PLEX Ag assay had a limit of detection (LOD) of 0.16 pg/ml and a cutoff of 0.32 pg/ml. Ag concentrations measured in clinical NP samples (collected in 3.0 ml of media) ranged from less than 160 fg/ml to 2.7 μg/ml. Log-transformed Ag concentrations correlated tightly with CT values. In 35 adult and 101 pediatric PCR-positive samples, the sensitivities were 91% (95% confidence interval, 77 to 98%) and 79% (70 to 87%), respectively. In samples with a CT of ≤35, the sensitivities were 100% (88 to 100%) and 96% (88 to 99%), respectively. In 50 adult and 40 pediatric PCR-negative specimens, the specificities were 100% (93 to 100%) and 98% (87 to 100%), respectively. Nucleocapsid concentrations in clinical NP samples span 8 orders of magnitude and correlate closely with RNA concentrations (CT values). The S-PLEX Ag assay showed 96 to 100% sensitivity in samples from children and adults with CT values of ≤35, and a specificity of 98 to 100%. These results clarify Ag concentration distributions in clinical samples, providing insight into the performance of Ag RDTs and offering a new approach to diagnosis of COVID-19.