We have previously shown that the serum parathyroid hormone (PTH) concentration in lactating (L) rats is not suppressed by high serum Ca²⁺ to the same extent as in nonmated (NM) rats. To investigate further Ca²⁺ regulation of PTH secretion, parathyroid cells from NM rats and rats in late pregnancy and at peak lactation were dispersed and incubated for 2 h in medium containing 0.52–2.05 mM Ca²⁺. Medium PTH was assayed with a homologous immunoradiometric assay (IRMA). At the two highest Ca²⁺ levels (1.81 and 2.05 mM), medium PTH was significantly higher (p = 0.031) for cells from L rats than for cells from NM rats. In contrast, significantly less (p < 0.001) PTH was secreted for the L group versus the NM group at medium Ca²⁺ values of 1.27 and 1.46 mM. Estimated set points for L and NM groups were 1.17 mM and 1.35 mM, respectively, corresponding closely to the prevailing serum Ca²⁺ for these two groups. Consistent with the present in vitro data, high serum PTH (>40 pg/ml) in L rats occurred only at serum Ca²⁺ values below 1.27 mM. Elevated serum PTH at lower serum Ca²⁺ levels was also seen in pregnant rats. Dispersed parathyroid cells from 20− to 21‐day pregnant rats secreted significantly more PTH (p = 0.028) than cells from NM rats at all Ca²⁺ levels tested (1.1–1.6 mM). In conclusion, the relationship between extracellular Ca²⁺ and PTH secretion is altered in rats during late pregnancy and at peak lactation, perhaps as part of the adaptation to the demands for calcium for pre‐ and postnatal growth.