Puwainaphycin C, a cardioactive agent which elicits a strong, positive inotropic effect in isolated mouse atria, is a novel chlorine-containing cyclic decapeptide from a Hawaiian terrestrial Anabaena sp. (strain BQ-16-1). Detailed spectral analysis of the intact molecule and an acid hydrolysis study show that puwainaphycin C possesses an unusual β-amino acid unit, vi., a 3-amino- 14-chloro-2-hydroxy-4-methylpalmityl residue, and nine known amino acid units. Using difference NOE spectroscopy and two-dimensional 13C-13C and carbon-detected 13C-15N correlation spectroscopy to sequence the 10 amino acid residues, the gross structure of puwainaphycin C has been shown to be cyclic(3-amino-14-chloro-2-hydroxy-4-methylpalmityl-valyl-2-aminobut-2( E)-enoyl-threonyl-threonyl-glutaminyl-glycyl-O-methylthreonyl-N-methylasparaginyl-prolyl). Puwainaphycin D, a closely related inactive compound, differs from puwainaphycin C in having a second valyl unit at the site occupied by Thr-2.