A comparison was made of the survival of 28 leptospiral serotypes in Fletcher semisolid medium and in the same medium containing a basal layer of Fletcher medium plus 0.7% of agar and 0.5% of activated animal charcoal. A year after culture, more motile leptospires were observed by microscope examination in the biphasic medium. Two years after culture, 4 serotypes grown in the biphasic medium and 11 in Fletcher medium did not show motile cells. Nineteen of the serotypes maintained in Fletcher medium and 25 in the biphasic medium for 2 years grew on subculture into Fletcher medium. Subcultures from the biphasic medium showed the characteristic leptospiral ring growth earlier during the incubation period.