The endoplasmic reticulum (ER) is the major compartment for the processing and quality control of newly synthesized proteins. Green fluorescent protein (GFP) was used as a noninvasive probe to determine the viscous properties of the aqueous lumen of the ER. GFP was targeted to the ER lumen of CHO cells by transient transfection with cDNA encoding GFP (S65T/F64L mutant) with a C-terminus KDEL retention sequence and upstream prolactin secretory sequence. Repeated laser illumination of a fixed 2-micrometers diameter spot resulted in complete bleaching of ER-associated GFP throughout the cell, indicating a continuous ER lumen. A residual amount (<1%) of GFP-KDEL was perinuclear and noncontiguous with the ER, presumably within a pre- or cis-Golgi compartment involved in KDEL-substrate retention. Quantitative spot photobleaching with a single brief bleach pulse indicated that GFP was fully mobile with a t1/2 for fluorescence recovery of 88 +/- 5 ms (SE; 60x lens) and 143 +/- 8 ms (40x). Fluorescence recovery was abolished by paraformaldehyde except for a small component of reversible photobleaching with t1/2 of 3 ms. For comparison, the t1/2 for photobleaching of GFP in cytoplasm was 14 +/- 2 ms (60x) and 24 +/- 1 ms (40x). Utilizing a mathematical model that accounted for ER reticular geometry, a GFP diffusion coefficient of 0.5-1 x 10(-7) cm2/s was computed, 9-18-fold less than that in water and 3-6-fold less than that in cytoplasm. By frequency-domain microfluorimetry, the GFP rotational correlation time was measured to be 39 +/- 8 ns, approximately 2-fold greater than that in water but comparable to that in the cytoplasm. Fluorescence recovery after photobleaching using a 40x lens was measured (at 23 degrees C unless otherwise indicated) for several potential effectors of ER structure and/or lumen environment: t1/2 values (in ms) were 143 +/- 8 (control), 100 +/- 13 (37 degrees C), 53 +/- 13 (brefeldin A), and 139 +/- 6 (dithiothreitol). These results indicate moderately slowed GFP diffusion in a continuous ER lumen.