Diffusion coefficients for the intercellular movement of fluorescent tracers have been measured in the epidermis of a larval beetle. Fluorescent tracer was injected into a cell and the spread of tracer from cell to cell in this monolayer was recorded by a TV camera. Fluorescence intensities were digitized from the TV images at successive times after the start of injection at various distances from the source by a microcomputer interfaced with a video analyzer. From the relationship between concentration (measured as light intensity), time and distance, an effective diffusion coefficient (De) is calculated for the tracer in the tissue. In newly ecdysed epidermis, De for carboxyfluorescein (CF) is 2.7 X 10(-7) cm2/s, and De for lissamine rhodamine B (LRB) is 1.2 X 10(-7) cm2/s, whereas in intermolt epidermis the De's for CF and LRB are 3.7 X 10(-7) and 1.2 X 10(-7) cm2/s, respectively. These diffusion coefficients are only an order of magnitude lower than their values in water. The ratio of De for the two tracers at these two stages of development differs from the ratio predicted in cytoplasm alone, with the movement of the slightly larger molecule (LRB) being impeded relative to that of the smaller molecule (CF). This suggests that the properties of the membrane channels amplify differences in the rates of movement of molecules of similar size. This may be important during cell patterning in development. De for CF was also monitored as junctional resistance was increased in the epidermis. During 30 min of exposure to 0.25 mM chlorpromazine, De dropped to 20% of its initial value of 5 X 10(-7) cm2/s, implying that the junctional membrane, rather than cytoplasm, is the major barrier to molecular diffusion among the cells.