Although it is well known that orthopedic complaints such as lumbago and shoulder pain can be reduced by hot spring bathing, the mechanism of such reduction is not yet fully known. Therefore, to clarify the mechanism, how the metabolism of glycosaminoglycans, which form connective tissue of articulations and cartilage, changes during hot spring bathing was investigated. Urine was sampled from those visitors at a hot spring who have orthopedic complaints such as lumbago but with no internal disease immediately before the session of hot spring bathing and at intervals of 1 week for a period of 5 week after starting hot spring bathing. The glycosaminoglycan fractions were separated from urine using the cetylpyridinium chloride precipitation and ethanol precipitation methods. Uronic acid contents were then estimated by the carbazole reaction method. The glycosaminoglycan levels in urine increased temporarily one week after starting hot spring bathing, then decreased to the level before starting bathing. The results of investigations using two-dimensional electrophoresis on cellulose acetate membranes revealed that chondroitin and chondroitin sulfate increased one week after beginning hot spring bathing. Because chondoritin is a progressive degradation product of chondroitin sulfate, the increase in chondroitin means that the metabolism of the chondroitin sulfate in the connective tissue was temporarily accelerated by hot spring bathing and was reflected as a reduction in complaints.