The folate receptor (FR) is a valuable therapeutic target that is highly expressed on a variety of cancers. The current development of folate-targeted cancer therapies has created the need for quantitating functional FRs in clinical specimens. In this article, we report on the creation of a highly sensitive radioactive binding method for quantitatively measuring FR expression in frozen tissue homogenates. Expression was positive in approximately 89% of human ovarian carcinomas but was negligible in both mucinous ovarian carcinomas and normal ovary. Expression was also significant in carcinomas of the kidney, endometrium, lung, breast, bladder, and pancreas. Normal tissues from humans and six different laboratory species were also analyzed; surprisingly, some interspecies variability in FR expression (especially in kidney, spleen, and lung tissue) was found. Interestingly, normal human lung tissue displayed high expression levels, whereas expression in normal lung of the other species was negligible. However, considering that folate-drug conjugates fail to accumulate in the lungs of patients, the consequence of this finding was not considered to be of clinical concern. Overall, this new methodology is reliable for determining functional FR expression levels in tissues, and it could possibly be a useful clinical test to determine patient candidacy for FR-targeted therapeutics.