This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to α1‐adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen‐coated polycarbonate membranes, or on collagen‐coated glass coverslips. Quin‐2 and fura‐2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin‐2, and 122 nM, as determined using fura‐2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10−5 M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin‐2 and fura‐2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180–300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin‐2 or fura‐2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell‐impermeant fura‐2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid‐phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+‐indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells “scrape‐loaded” with fura‐2 revealed a four‐fold increase in [Ca2+]i concentration following stimulation with phenylephrine.