A comparison of TMP-pNP activation by ENPP-1versus alkaline phosphatase (ALP) reveals a complete lack of selectivity.Removal of the self-immolative spacer from probe CL-ENPP-1 resulted in a new chemiluminescent probe, CL-ENPP-2, with an 18.4-fold increase in selectivity for ENPP-1 over ALP.The ability of probe CL-ENPP-2 to detect ENPP-1 activityin mammalian cells was assessed using the human breast cancer cellline MDA-MB-231. Here, ENPP1 is linked to breast cancer.