The present study evaluated the use of a small animal total body software of dual energy x-ray absorptiometry (DXA) in the assessment of total body and regional bone mineral content (BMC) and bone mineral density (BMD) in small rats. Twenty-three rats, with weights ranging from 146 to 246 g, were included in the study. All were scanned using the same software version and same scan procedure (speed and scanned area). Total body BMD, BMC, and body weight were measured by DXA in each rat. Femoral BMC and BMD were analyzed by using regional analysis facilities. The repeatability (precision) of this software version was assessed prior to the study and the coefficients of variation (CV) were 2.9% for total body BMC, 0.8% for total body BMD, 1.2% for body weight, and 2.2% for mean femoral BMD. DXA measurements were compared with the measurements obtained by using established standards, namely weight and bone ash content. Total body ash content and femoral ash content were measured separately in all rats. There was a strong linear correlation between BMC and ash content in total body (r2 = 0.98, p = 0.0001) and in femur (r2 = 0.94, p = 0.0001). There was also an excellent linear association between body weight measured by DXA and scale weight (r2 = 0.99, p = 0.0001). We conclude that this software version is suitable for study on small animals and is a useful tool for assessment of regional as well as total body bone mineral status.