The purposes of this study were: (a) to measure the translational mobility of a small solute in cell cytoplasm; (b) to define quantitatively the factors that determine solute translation; and (c) to compare and contrast solute rotation and translation. A small fluorescent probe, 2,7-bis-(2-carboxyethyl)-5-(and 6-)-carboxyfluorescein (BCECF), was introduced into the cytoplasm of Swiss 3T3 fibroblasts. BCECF translation was measured by fluorescence recovery after photo-bleaching; rotation was measured by Fourier transform polarization microscopy. Diffusion coefficients relative to those in water (D/D0) were determined by comparing mobility in cytoplasm with mobility in standard solutions of known viscosity. At isosmotic cell volume, the relative diffusion coefficients for BCECF translation and rotation in cytoplasm were 0.27 +/- 0.01 (SEM, n = 24, 23 degrees C) and 0.78 +/- 0.03 (n = 4), respectively. As cell volume increased from 0.33 to 2 times isosmotic volume, the relative translational diffusion coefficient increased from 0.047 to 0.32, while the relative rotational diffusion coefficient remained constant. The factors determining BCECF translation were evaluated by comparing rotation and translation in cytoplasm, and in artificial solutions containing dextrans (mobile barriers) and agarose gels (immobile barriers). It was concluded that the hindrance of BCECF translation in cytoplasm could be quantitatively attributed to three independent factors: (a) fluid-phase cytoplasmic viscosity is 28% greater than the viscosity of water (factor 1 = 0.78); (b) 19% of BCECF is transiently bound to intracellular components of low mobility (factor 2 = 0.81); and most importantly, (c) translation of unbound BCECF is hindered 2.5-fold by collisions with cell solids comprising 13% of isosmotic cell volume (factor 3 = 0.40). The product of the 3 factors is 0.25 +/- 0.03, in good agreement with the measured D/D0 of 0.27 +/- 0.01. These results provide the first measurement of the translational mobility of a small solute in cell cytoplasm and define quantitatively the factors that slow solute translation.