Polyethylene glycol (PEG) addition can prolong the pharmacokinetic and pharmacodynamic actions of a bioactive peptide in vivo, in part by impeding rates of glomerular filtration. For parathyroid hormone (PTH) peptides, pegylation could help in exploring the actions of the hormone in the kidney; e.g., in dissecting the relative roles that filtered versus blood‐borne PTH play in regulating phosphate transport. It could also lead to potential alternate forms of treatment for hypoparathyroidism. We thus synthesized the fluorescent pegylated PTH derivative [Lys¹³(tetramethylrhodamine {TMR}), Cys³⁵(PEG‐20,000 Da)]PTH(1‐35) (PEG‐PTH^TMR) and its non‐pegylated counterpart [Lys¹³(TMR), Cys³⁵]PTH(1‐35) (PTH^TMR) and assessed their properties in cells and in mice. In PTHR1‐expressing HEK‐293 cells, PEG‐PTH^TMR and PTH^TMR exhibited similar potencies for inducing cAMP signaling, whereas when injected into mice, the pegylated analog persisted much longer in the circulation (>24 hours versus ∼ 1 hour) and induced markedly more prolonged calcemic and phosphaturic responses than did the non‐pegylated control. Fluorescence microscopy analysis of kidney sections obtained from the injected mice revealed much less PEG‐PTH^TMR than PTH^TMR on the luminal brush‐border surfaces of renal proximal tubule cells (PTCs), on which PTH regulates phosphate transporter function, whereas immunostained phosphorylated PKA substrate, a marker of cAMP signaling, was increased to similar extents for the two ligands and for each, was localized to the basolateral portion of the PTCs. Pegylation of a bioactive PTH peptide thus led to prolonged pharmacokinetic/pharmacodynamic properties in vivo, as well as to new in vivo data that support a prominent role for PTH action at basolateral surfaces of renal proximal tubule cells. © 2016 American Society for Bone and Mineral Research.