NCS1 (Frequenin) is a member of neuronal calcium sensor (NCS) family. This globular protein contains four EF-hand motifs and an amino-terminal myristylation sequence. Pb2+has been widely viewed as a health concern for its anthropogenic toxicity. It has been observed that Pb2+ modulates interactions between calcium-binding protein calmodulin (CaM) and its binding partners through binding in the EF-hands of CaM and it may play a significant role in Pb2+-induced neurotoxicity. Mn is a highly abundant metal in nature which has been found in human brain and associated with the several diseases like multiple sclerosis, Alzheimer's and Parkinson's disease and is considered as potential toxic element. Previous studies from our group showed Pb2+ and Cd2+ binding to DREAM protein that can contribute to Pb2+/Cd2+ induced neurotoxicity. Here we showed that Pb2+ binds to EF-hands in apo-NCS1with a high affinity (Kd = 7.0±1.5 nM) whereas Cd2+ did not show any binding with NCS1. For the first time we investigated the Mn2+ association to NCS1 protein. This cation binds with an apparent equilibrium dissociation constant of 340±67 µM. Pb2+ and Mn2+binding trigger secondary and tertiary structural rearrangements of NCS1 that are analogous to those observed for Ca2+ association. In addition, Pb2+ as well as Mn2+ promote NCS1 interaction with hydrophobic molecule 1,8-ANS. Interestingly,Ca2+NCS1:1,8-ANS complex exhibits a co-operative binding of Pb2+ with Kd = 103±23 µM whereas Mn2+ showed apparent Kd value 924±185 µM with Ca2+NCS1:1,8-ANS complex. These results suggest allosteric interactions between the 1,8-ANS binding site and Pb2+ binding EF-hands. Data provided here points towards ability of EF-hands in NCS-1 to interact with various metals and a possible role of NCS1 in Pb2+/Mn2+ neurotoxicity.