To estimate total cleft length per unit surface area, previous studies assumed that endothelial cell clefts were oriented randomly with respect to the axis of the microvessel (Bundgaard and Frokjaer-Jensen, Microvasc. Res. 23, 1-30, 1982). In the present study, silver precipitation along the intercellular clefts of capillary endothelium ('silver lines') in the frog mesentery allowed observation of cleft orientation as well as estimation of cell shape, cell area (C(A)), and cleft length per unit area (C(L)). In all vessels, the endothelial cells were highly elongated polygons, usually hexagonal, which were oriented along the vessel axis. The clefts were highly oriented with a preferred orientation which was parallel to the vessel axis. Clefts exhibited very little local meandering, with a contour length only 7% greater than endpoint-to-endpoint length. Therefore, the assumption of random cleft orientation was not valid, and its use yielded an overestimate of C(L). New estimates of C(L) were 0.16, 0.12, and 0.12 μm-1 for arterial, true, and venous capillaries, respectively. Cell lengths (mean ± SD, n) were 135 μm (± 28, 79) in arterial capillaries, 98 μm (± 28, 19) in true capillaries, and 139 μm (± 20, 21) in venous capillaries. © 1993 Academic Press, Inc.