To study possible influences of a mild increase in serum-ionized calcium concentration that is seen during daily life on circulating parathyroid hormone (PTH) immunoheterogeneity, we used sequence-specific PTH assays to determine serum intact PTH, C-terminal PTH and N-terminal PTH following oral calcium (1.5 g) in healthy subjects (n = 7). This was also performed in patients with primary hyperparathyroidism (pHPT; n = 10) to see if their regulation of circulating PTH molecular forms is normal. Compared to healthy subjects, the patients were hypercalcemic (p < 0.05) and had higher levels of PTH in all three assays (p < 0.001). Following the oral calcium load, serum-ionized calcium increased by 0.08 ± 0.03 mmol/l in the patients and by 0.07 ± 0.03 mmol/l in the healthy subjects after 90 min, whereas serum intact PTH, C-terminal PTH and N-terminal PTH were reduced, both in the healthy subjects and in the patients. Suppression by calcium of both intact PTH and C-terminal PTH were impaired in the patients (p < 0.05 and p < 0.001), whereas suppression of N-terminal PTH was normal. Furthermore, the C/i and N/i ratios were higher at the highest calcium concentration achieved after calcium intake in the healthy subjects than in the basal state in the patients (p < 0.05), in spite of the larger degree of hypercalcemia in the latter (1.40 ± 0.06 vs. 1.31 ± 0.02 mmol/l; p < 0.05). Thus, (1) a minor increase in serum-ionized calcium that is seen during daily life alters the relative circulating concentrations of PTH versus its fragments; (2) the impaired sensitivity to calcium in pHPT is not evident for the suppression of N-terminal PTH, and (3) pHPT is accompanied by altered immunoheterogeneity of circulating PTH.