The ratio of the numbers of atypical colonies (AC) to typical total coliform colonies (TC) has been used successfully to identify predominant sources of fecal contamination, predict the presence or absence of enteric viruses, estimate age, and examine changes in land use and sewage collection within urban, agricultural, and mixed-use surface watersheds. We conducted a laboratory aging experiment approximating karst field conditions along with the extensive monitoring of a well-characterized karst spring during December 2002–March 2004 and February–October 2005. The experiment showed that cooler temperatures retarded the expected change in the AC/TC ratio in comparison with warmer conditions analogous to surface water during late spring, summer, and early autumn. A statistically significant decrease in the AC/TC ratio was observed for monitoring data collected during 2005 relative to earlier data. This decrease is presumed to be a result of accidental sewage spills associated with construction in the vicinity of the spring. These investigations determined that the AC/TC ratio, in combination with FC data, can be a useful tool for delineating “hot spots” of fresh fecal inputs within a karst groundwater basin.