Rock varnish occurs in virtually all environments, most commonly in arid and semi-arid climates, including Antarctica. Rock varnish consists of thin layers of intimately mixed aeolian and chemical sediments often showing botryoidal and more rarely stromatolite-like morphologies. Typical rock varnish samples collected at Twin Peak Mountain Park, near Phoenix, Arizona, consist of abundant quartz, with plagioclase, illite and a mixed layer, Fe-clay mineral, probably corrensite. EDS, SEM (BSE) and TEM analyses revealed that the typical Mn, Fe minerals occur as minute particles; some of these particles and other mineral grains are attached to filaments. XRD and electron diffraction showed that the Mn.Fe-bearing particles are poorly crystalline. The filaments, based on morphological criteria, are virtually indistinguishable from fungal filaments. Most filaments are fragments, probably broken by scraping during sample collection. Coccoid and rod-shaped forms, resembling cyanobacteria and other bacteria, respectively, are also present. Unlike definitive minerals, these filaments disintegrated in the concentrated energy of the SEM electron beam at the instrumental and experimental conditions used. In addition, no filamentous, rod-shaped or coccoid forms were observed in samples hydrolysed with 6 N HCl for 24 h at 100°C. Bacteria and fungi in powdered rock varnish were cultured on four media, incubated aerobically in the dark at 25°C. The culture media yielded dense growths of spore-forming bacteria and filamentous fungi. One fungus and two Bacillus isolates oxidized and concentrated manganese. Control experiments revealed that fungi and bacteria are present on and below the surfaces of rock varnish. Free and hydrolysed, peptide/protein-bound amino acids were identified in the rock varnish. Amino acids showed virtually no racemization with the exception of D/L asp = 0.1. Relatively high molecular weight humic matter was also separated from the rock varnish. High-resolution mass spectrometry revealed non-hydrocarbon moieties, similar to a Suwannee River (FL) humic acid standard. Micro-organisms and their original biochemical compounds do not seem to be preserved for long in the accreting varnish layer. The studies showed that the filaments helped to trap mineral particles of rock varnish, and that bacteria and fungi abetted Mn concentration. Some structures in the layers of rock varnish resemble stromatolites and present definitions would allow them to be termed as such.