As a study organism, annual killifish (Austrofundulus limnaeus) provide a well suited study system for examining the effects of environmental temperature fluctuations at the cellular level. A. limnaeus persist in the harsh high desert climate of the Maracaibo Basin, Venezuela where they live in small, ephemeral freshwater pools. Temperatures in these waters can vary as much as 20 degrees C daily and reach maximums of over 40 degrees C due to the semi-arid climate. Previous cDNA microarray studies on killifish revealed the mRNA pattern for High Mobility Group Protein 1 (HMGB1) to be strongly affected by temperature perturbations. Specifically, peaks in hmgb1 transcript abundance were negatively correlated with temperature during temperature cycling, and experienced over a 10 fold difference in expression in response to the temperature cycle. Using the same temperature cycling experimental setup, this study's aim was three-fold: (1) to characterize the total amount of HMGB1 protein in adult male killifish livers, (2) to describe the subcellular localization of the HMGB1 protein in adult male killifish livers and (3) to sequence the 5' upstream region of the hmgb1 gene to identify possible stress responsive elements. We detected no significant difference in total HMGB1 protein levels as a consequence of temperature cycling. The data for subcellular localization of HGMB1 protein do not support a strong change in subcellular localization of the protein in response to temperature cycling; most of the HMGB1 protein is found in the cytoplasmic compartment in liver tissue. Although overall patterns of subcellular localization did not change significantly, we found a significant difference between nuclear HMGB1 protein levels in temperature cycled fish versus control (constant temperature) fish. This could suggest a muting of the natural translocation of HMGB1 into the nucleus observed in control fish at around 9:00 at night. Finally, the upstream region of the hgmb1 gene does reveal a number of putative stress responsive transcription factor binding sites.