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Scientists vigorously debate the degree to which rock varnish is formed through the actions of microorganisms. To investigate this enigma, we utilized a three-pronged approach that combined (1) culture-independent molecular methods to characterize bacterial communities associated with varnish that coats the rhyolitic volcanic rocks of Black Canyon, New Mexico, and rocks with no visible varnish; (2) culturing of varnish in media supplemented with reduced forms of manganese and/or iron and no or low amounts of carbon to isolate bacteria capable of precipitating iron and/or manganese oxides; and (3) scanning electron microscopy (SEM) of varnish and nearby rock that lacks macroscopically visible varnish. Our culture-independent studies revealed significant differences between varnish and nonvarnish communities. Chloroflexi and Ktedobacteria dominated one varnish site, while the other varnish site was dominated by Cyanobacteria. The nonvarnish sites were dominated by Actinobacteria and, to a lesser extent, Cyanobacteria and were the only samples to contain Deinococcus-Thermus sequences. Approximately 65% of varnish cultures produced visible manganese precipitates. Most culture isolates were not closely related to known manganese oxidizers, with the exception of Bacillus spp. SEM revealed microbial morphologies and two types of varnish morphologies: (1) relatively smooth layers and (2) patches of botryoidal pinnacles, which were often associated with increased manganese concentrations. "Bare" rock showed evidence of incipient varnish. These results have important implications for the detection of life on extraterrestrial planets such as Mars, where putative varnish coatings have been observed, and represent some of the first culture-independent characterizations of varnish communities.