Macrotetrolides isolated from a new producer, Streptomyces globisporus, were identified as nonactin, monactin, dinactin and trinactin. Spectroscopic characterization of these compounds was extended by 13NMR spectra. Chemical ionization with ammonia as reactive gas was proposed for mass-spectroscopic characterization of their mixtures. Their biological activity was confirmed by using larvae of the Colorado potato beetle (Leptinotarsa decemlineata) as a new test model.