The transport of newly synthesized cholesterol from its site of synthesis, the endoplasmic reticulum, to the plasma membrane was studied in CaCo-2 cells. The appearance of newly synthesized cholesterol on the cell surface was rapid. By 30 min, 50% of the total labeled cholesterol was observed in the plasma membrane. The arrival of cholesterol at the plasma membrane was independent of new protein synthesis, a functional Golgi apparatus, or microtubular function. Progesterone, verapamil, and trifluoperazine, inhibitors of p-glycoprotein which are known to inhibit cholesterol transport from the plasma membrane to the endoplasmic reticulum, reduced the amount of newly synthesized cholesterol reaching the plasma membrane. The p-glycoprotein inhibitors, however, caused the accumulation of sterol intermediates in the plasma membrane, suggesting that sterol trafficking to the plasma membrane remained intact, but that trafficking from the plasma membrane to the endoplasmic reticulum was disrupted. In contrast, nigericin, another potent inhibitor of cholesterol movement from the plasma membrane to the endoplasmic reticulum, did not alter the transport of newly synthesized cholesterol to the plasma membrane. Moreover, promoting cholesterol transport from the plasma membrane to the endoplasmic reticulum by sphingomyelin hydrolysis or by micellar cholesterol influx did not alter the percent of newly synthesized cholesterol transported to the plasma membrane. Likewise, preventing plasma membrane cholesterol from reaching the endoplasmic reticulum by incubating cells with lysophosphatidylcholine, filipin, or digitonin did not alter the arrival of newly synthesized cholesterol to the plasma membrane. The results suggest that the amount of cholesterol moving to the plasma membrane from the endoplasmic reticulum is constitutive and regulated at the level of cholesterol synthesis and not at the level of the transport process. The pathways of cholesterol transport to and from the plasma membrane are distinct.