The volume occupied by the extracellular space has been investigated in six types of voluntary muscles: sartorius (frog), semitendinosus (frog), tibialis anticus longus (frog), iliofibularis (frog), rectus abdominis (frog), and diaphragm (rat). With the aid of four types of probe material, three of which are conventionally employed (inulin, sorbitol, sucrose) and one of which is newly introduced (poly-L-glutamate), and a different experimental method, we have demonstrated that the "true" extracellular space of frog sartorius, semitendinosus, tibialis anticus longus, and iliofibularis muscle and of rat diaphragm muscle is equal to, or probably less than, 8-9 % (v/w) of the tissue. The frog rectus muscle shows a somewhat higher ceiling value of 14 %.Living tissue consists of cells and "extracellular space." A precise knowledge of the percentage of extracellular space is essential if one is to obtain meaningful values for the concentrations of solutes or water within the cell. In the case of solutes like Na+ ion and glucose, which are usually found at lower concentrations within the cell than in the extracellular fluid, an erroneous value for the extracellular space may lead to the conclusion that these solutes are altogether absent in the cell, while in truth they are present in sizable concentrations (or vice versa). Yet published values for the extracellular space vary widely. Thus, for frog sartorius muscle the proportion of extracellular space given in the literature varies from 13% of the tissue (1-4) to as high as 35% (5-9). With this degree of uncertainty, meaningful interpretation of quantitative data on "glucose transport" or "sodium transport" in muscle tissues, as examples, is virtually impossible. Such problems are of fundamental importance in understanding the behavior of the living cell.In this communication we present results of investigations of the extracellular space of several types of voluntary muscles of amphibian and mammalian origin. Using new techniques, we have found the extracellular spaces of these tissues to be surprisingly low and constant.