The use of stable isotopes to label phylogenetically informative biomolecules (phospholipid fatty acids, DNA, or RNA), typically referred to as stable isotope probing (SIP) has the potential of providing definitive evidence that a detected population is active in a specific process, if that process results in assimilation of C or N into cellular constituents. Application of SIP techniques to unsaturated soils, however, should be treated with the same caution as other attempts to address function in soils. Of particular concern is the issue of bioavailability of the test substance, a problem that is often neglected in ecology studies. Many substrates are subject to sorption in soil, and introduction of a labeled test substance throughout pore space in unsaturated soil is not instantaneous. Considerable time may be required for added material to become well distributed among soil pores. Inasmuch as a very high rate of label incorporation is required for nucleic acid based SIP (as much as 20% of DNA may need to be labeled in the case of 15N-SIP), bioavailability limitations may preclude accumulating sufficient label to achieve centrifugal separation of enriched from un-enriched nucleic acids. Herein we report experiments demonstrating evidence for limited bioavailability in the degradation of several organic contaminants and present labeling requirements we have observed for successful DNA-SIP. We conclude that SIP experiments with unsaturated soils can benefit by including an assessment of substrate bioavailability to facilitate interpreting results. Monitoring/predicting soil solution phase or labile material may provide insight into the impact of sorption on bioavailability at the bulk scale. Other approaches will be necessary to assess the physical availability of solution phase material at the scale of habitable pores. This may require additional methods development.