We investigated the relative contribution of the major factors regulating calcium homeostasis in determining the circulating levels of PTH. We studied 137 males and 125 females who were healthy volunteers. Circulating PTH levels were determined by three different immunoradiometric assays (IRMA). The first one (PTH Sorin, PTH S) utilizes two affinity-purified polyclonal antibodies directed against the 1-34 and 39-84 sequence of the hormone. The two other IRMA share polyclonal anti-PTH (39-84) antibodies. The first assay (PTH Whole, PTH W) utilizes a second polyclonal antibody, directed against the 1-4 amino acid sequence. The second assay (PTH Total, PTH T) utilizes a second antibody specific for the 7-34 region. Concentrations of PTH fragments lacking the initial amino acid sequence (PTH N-truncated, PTH N-t) were determined by the difference of values between PTH T and PTH W. Vitamin D was the main explicative variable almost in every multiple linear regression model, both considering the group as a whole (PTH S: R2 = 0.238, P < 0.0001; PTH W: R2 = 0.08, P < 0.001; PTH T: R2 = 0.145, P < 0.0001; PTH N-t: R2 = 0.081, P < 0.009) and when considering men and women separately. In subjects with vitamin D insufficiency (n = 53) [25(OH)D < 30 nmol/l], mean serum levels of parathyroid hormone were significantly higher (P < 0.001) than those in subjects of similar age with normal vitamin status (n = 209) with all the assays employed. This study demonstrates the central role of 25(OH)D in regulating PTH secretion in physiological conditions.