Cyanobacteria from a diversity of marine and freshwater habitats are known to produce neurotoxic secondary metabolites. Herein, we describe the complete stereostructure, synthesis, and biological properties of kalkitoxin (1), a novel neurotoxic lipopeptide from a Caribbean collection of Lyngbya majuscula. The organic extract of this L. majuscula exhibited potent brine shrimp and fish toxicity. Using these assays, the toxic metabolite kalkitoxin (1) was isolated by sequential silica gel VLC, CC, and normal-phase HPLC (12.8 mg, 0.3% of extract). Subsequently, bioassay-guided fractionation using a primary cell culture of rat neurons in a microphysiometer or inhibition of IL-1β stimulation of sPLA2 in hepatocarcinoma cells led to re-isolation of 1 in small yield from various Caribbean collections of L. majuscula. Kalkitoxin (1) analyzed for C21H38N2OS indicated four degrees of unsaturation; from 13C NMR analysis in DMSO-d6 two were due to double bonds, one to a carbonyl group, and the remaining one to a ring system. Data from E.COSY, HSQC, and a modified HSQMBC experiments in benzene-d6 allowed deduction of six partial structures for 1. The C3 stereochemistry of kalkitoxin was determined by Marfey's analysis, yielding L-cysteic acid and defining C3 as R. The relative stereochemistry of the three chiral centers within the aliphatic chain (C7, C8, C10) was determined using the J-based configuration analysis method, supporting a 7R*, 8S*, 10S* relative stereochemistry. To determine the absolute stereochemistry of natural kalkitoxin, kalkitoxins having all possible configurations were synthesized; (3R,7R,8S,10S,2′R)-kalkitoxin was found to be identical with the natural substance 1. Natural (+)-kalkitoxin was strongly ichthyotoxic to the common goldfish (LC50 700 nM), potently brine shrimp toxic (Artemia salina, LC50 170 nM), and potently inhibited cell division in a fertilized sea urchin embryo assay (IC50 ~25 nM). Synthetic (+)-kalkitoxin was equally potent in the brine shrimp assay (LC50 170 nM), while synthetic (-)-kalkitoxin was relatively inactive (LC50 9300 nM). In a primary cell culture of rat neurons, natural kalkitoxin displayed an exceptional level of neurotoxicity (LC50 3.86 nM), and its effects were inhibitable with NMDA receptor antagonists. Additionally, natural kalkitoxin is highly active in an inflammatory disease model which measures IL-1β-induced sPLA2 secretion from HepG2 cells (IC50 27 nM). Preliminary evidence suggests that kalkitoxin is an exquisitely potent blocker of the voltage sensitive Na+ channel in mouse neuro-2a cells (EC50 of 1 = 1 nM; EC50 of saxitoxin = 8 nM). Consistent with many known cyanobacterial metabolites, kalkitoxin appears to derive from a mixed polyketide/nonribosomyl peptide synthetase pathway.