Geographic venom samples of Crotalus viridis viridis were obtained from South Dakota, Wyoming, Colorado, Oklahoma, Texas, New Mexico, and Arizona. From these samples, the phospholipases A(2) (PLA(2)s) were purified and their N-terminal sequences, precise masses, and in vitro enzymatic activities were determined. We purified two to four distinct acidic PLA(2)s from each sample; some of them displayed different inhibition specificities toward mammalian platelets. One of the acidic PLA(2)s induced edema, but had no anti-platelet activity. There was also a common basic PLA(2) myotoxin in all the samples. We have cloned five acidic PLA(2)s and several hybrid-like nonexpressing PLA(2)s. Molecular masses and N-terminal sequences of the purified PLA(2)s were matched with those deduced from the cDNA sequences, and the complete amino acid sequences of five novel acidic PLA(2)s were thus solved. They share 78% or greater sequence identity, and a cladogram based on the sequences of many venom acidic PLA(2)s of New World pit vipers revealed at least two subtypes. The results contribute to a better understanding of the ecogenetic adaptation of rattlesnakes and the structure-activity relationships and evolution of the acidic PLA(2)s in pit viper venom.