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Andragulla Cave is 2 m high, 6 m wide, and 2 m long (deep). Its lack of depth means that it is not isolated from external influences, and the microclimate is very similar to that experienced externally. The common stress factors on the growth of cyanobacterial communities on walls inside of Andragulla Cave include excess light, dryness, lack of nutrients, and cold temperatures. Nitrogen fixation, photon flux, relative humidity, and temperature in Andragulla Cave were measured hourly over 24 hours in winter. Nitrogen fixation by the reddish-brown mat formed by both cyanobacteria Scytonema mirabile and Gloeocapsa sanguinea in cave-like environments was measured in situ for the first time by acetylene reduction. The mat-specific rates (1.6 to 7.5 nmol C(2)H(4) m(-2) s(-1)) were lower compared to published values from laboratory measurements of rehydrated samples from different environments. Daytime fixation was slightly higher than nighttime fixation, where nitrogen fixation by Gloeocapsa sanguinea played an important role. The most influential parameters for environmental nitrogen fixation in Andragulla Cave were photon flux for daytime fixation, temperature for nighttime fixation, and relative humidity for both. Nitrogen fixation by cyanobacteria may be significant in these N-poor ecosystems.