The entomopathogenic fungus Metarhizium anisopliae is generally considered to be mesophylic, although some isolates have the ability to grow at 8 °C. In two cold-active isolates (DAT-1 from Tasmania and CLB2-1vi from Ontario, Canada) and one non-cold-active isolate (Ma 2575 from South Carolina), we (1) analyzed cold-induced intracellular proteins, using two-dimensional (2D) gel electrophoresis, and (2) analyzed cold-active transcripts, using a polymerase chain reaction (PCR) based subtractive hybridization technique. Protein differences, as observed by 2D gel electrophoresis, were observed in cold active isolates grown at 8 ° when compared with 25 °, but such differences were not observed in the non-cold active isolate. Fungi were also subjected to various stresses (45 °, pH, and salinity) to determine how proteins induced under these conditions compared to proteins induced under low temperature growth. Proteins induced were either specific to cold-active growth or were general stress proteins and differences were also observed in 2D patterns between the two cold-active isolates. Transcripts upregulated during growth at 8 ° and isolated by a PCR based subtractive hybridization technique also differed between the two cold-active isolates. Analysis of the transcripts showed several novel sequences but also included transcripts with similarities to actin, NADPH quinone oxidoreductase, a thiamine biosynthesis protein and a yeast-like membrane protein. The potential role of these proteins in cold-active growth is discussed.