Aedes vexans (Diptera: Culicidae) larvae collected from ephemeral lentic habitats in the Haskell-Baker Wetlands, south of Lawrence, Kansas, were infested with a symbiotic fungus. Two isolates of Smittium culisetae, KS-108-02 and KS-108-06, were axenically cultured from the hindguts of the mosquito larvae. Physiological studies were conducted on isolate KS-108-02 to investigate conditions suitable for successful development of the fungus. The effects of temperature, pH, and different carbon and nitrogen sources on the growth of this new isolate were studied in vitro. Growth was measured as mycelium dry weight for all studies. Asexual trichospore production was monitored in the temperature, pH, and nitrogen source studies. Higher temperatures of 30 C and 24 C produced higher growth rates and trichospore production in shorter periods of time compared to temperatures of 18°C and 12°C. Media with lower pH produced greater growth than media with higher pH. Trichospore production was highest at pH 5.0, reaching an average of 1.21 × 10⁵ trichospores/ml, but above pH 5.0 trichospore production decreased. Glucose produced the greatest amount of growth followed by maltose, fructose, and soluble starch when the isolate was tested with various carbon sources. When tryptone concentrations in the culture medium were reduced to 0.1% in the nitrogen-source study, isolate KS-108-02 exhibited some reciprocal effects between growth and sporulation, wherein nitrogen sources that produced higher mycelium dry weight yielded lower sporulation and vice versa. Results of this study differed slightly from previous studies conducted on S. culisetae.