Mojave toxin (MT) was detected in five of 25 Crotalus helleri (Southern Pacific rattlesnake) sampled using anti-MT antibodies and nucleotide sequence analysis. All of the venoms that were positive for MT were collected from Mt San Jacinto in Riverside Co., California. Since this population is geographically isolated from C. scutulatus scutulatus (Mojave rattlesnake), it is unlikely that this finding is due to recent hybridization. MT concentration differences between C. helleri and C. s. scutulatus reflected the presence of 'isoforms' of the toxin in the venom. Whereas C. s. scutulatus generally has several isoforms of the toxin (detected by Western blotting), only one 'isoform' that focused at pI 5.1 was detected in C. helleri. Both acidic and basic subunits of MT sequences were obtained from C. helleri DNA with primers specific for MT, but only from snakes that had MT in their venom. The sequence identity of the C. helleri acidic subunit to the C. s. scutulatus subunit was 84.9%, whereas the sequence identity of the C. helleri basic subunit was 97% to the C. s. scutulatus basic subunit. Using casein, fibrin, and hide powder azure as substrates, assays for proteolytic activity suggested that C. helleri possesses several different types of metalloproteinases in their venom. However, proteolytic activity was not detected, or present in reduced amounts, in specimens having MT. Clinical neurotoxicity following envenomation by certain populations of C. helleri may be due to MT.