Previously, we have used the classical approach to examine intracellular calcium stores in human foreskin fibroblasts (HSWP) cells. In this classical protocol cells are first permeabilized and then allowed to fill their Ca 2 + reservoirs with 4 5 Ca 2 + in the presence of ATP. In this paper we present an alternative method to examine intracellular calcium pools. In this alternate protocol, whole cells are loaded to isotopic steady-state with 4 5 Ca 2 + and then permeabilized using digitonin. Comparison of the Ca 2 + content of cells treated with these two methodologies reveals that cells treated with the alternate protocol have a Ca 2 + content 3 orders of magnitude higher than those treated with the classical protocol. Using this alternative technique we demonstrate that there are 3 intracellular calcium pools in HSWP cells. These pools are: (1) an IP 3 -sensitive, thapsigargin-sensitive Ca 2 + pool; (ii) an IP 3 -insensitive, thapsigargin-sensitive Ca 2 + pool; and (iii) an ionomycin sensitive Ca 2 + pool. The relationship between the Ca 2 + pool mobilized by BK treatment and by IP 3 treatment is also explored. Microinjection data shown here suggest that IP 3 can mobilize all of the intracellular Ca 2 + mobilized by BK. However, in the permeabilized system BK pretreatment followed by IP 3 treatment can release more Ca 2 + than can be release by IP 3 treatment alone. We suggest one plausible explanation for this observation: when cells are treated using the alternative permeabilization protocol, communication occurs between an IP 3 -sensitive and an IP 3 -insensitive calcium pool. Thus BK pretreatment would empty the IP 3 -sensitive Ca 2 + pool. This pool would subsequently be refilled with Ca 2 + from a previously untapped, IP 3 -insensitive, Ca 2 + reservoir and more Ca 2 + would be available for subsequent release by IP3 treatment.