It is well established that renal proximal tubule (RPT) cells grown under standard in vitro conditions attenuate many of their in vivo properties and functions. Thus, the study of renal stress response mechanisms requires an appropriate cell culture model. In the present study, we compared the heat stress (10 min, 45°C) response of freshly isolated RPT cells with that of RPT cells grown in vitro for 6 days under two different culture conditions: (1) SHAKE conditions, where oxygen levels and physiological functions are maintained via continuous media motion [Nowak G, Schnellmann RG: Am J Physiol 1996;271:C2072–2080] and (2) STILL conditions, involving standard cell culture which leads to partial hypoxia and a marked reduction in physiological functions. The freshly isolated RPT cells progressively synthesized heat shock proteins (HSPs) and stress glycoproteins (SGs) during a 3-hour culture period in vitro. Under these conditions, heat stress did not further increase HSP and SG synthesis. In RPT cells grown under SHAKE conditions, HSP70 synthesis was detected 1 h after heat stress and decreased below detection by 3 h. In contrast, the uptake of radiolabeled mannose into (glycoprotein) GP62 (M<sub>r</sub> 62,000), GP50, and GP38 was observed in control SHAKE cultures and was not further increased after heat stress. These results are consistent with immunohistochemistry studies, where similar changes in HSP70 and GP50 expression were noted. RPT cells grown under STILL conditions showed both increased synthesis of HSP70 and increased glycosylation of GP62, GP50, and GP38 as early as 1 h after heat stress, but in contrast to SHAKE conditions, this heat-induced stress response further intensified at 3 h after heat stress. By 7 h after heating, HSP synthesis returned to control levels, while glycosylation of GP62 and GP50 remained elevated. Based on our results, we conclude that freshly isolated RPT cells exhibit a stress response that may be caused by acute cell isolation/culture stress. While this stress response unfolds, freshly isolated RPT cells appear unable to respond to additional heat stress. RPT cells grown under SHAKE and STILL conditions exhibit high rates of SG glycosylation, especially that of GP62, possibly reflecting a ‘stress’ condition associated with growth on plastic substrate. Concurrently, RPT cells from STILL cultures show a higher capacity for responding to acute heat stress than SHAKE cultures, evidenced by the transiently increased HSP synthetic rates. The interpretation of the renal stress response capacity, therefore, must be linked to a specific culture condition.