The effect of D-cycloserine on the establishment of twist states in Bacillus subtilis macrofibers was examined. Macrofibers produced in the presence of the drug differed in twist compared with those produced in its absence. The degree of twist alteration was dependent on the concentration of D-cycloserine in the growth medium. Macrofibers of different twist states representative of the entire twist spectrum from tight left-handedness to tight right-handedness were produced in strains FJ7 and C6D in four different ways: by control of the concentration of D-alanine, magnesium sulfate, or ammonium sulfate in the growth medium or by control of the growth temperature. The structures so produced were used to determine the effect of D-cycloserine on twist establishment starting from different twist states throughout the twist spectrum. In all but one case, twist resulting from growth in the presence of D-cycloserine was further towards the left-hand end of the twist spectrum than that produced in its absence, the exception being the unusual left-handed twist states produced in strains C6D and the closely related RHX 11S at high D-alanine concentrations described here. Studies of the interaction between D-cycloserine and D-alanine both used alone and used independently with the other twist-modifying systems (temperature, magnesium sulfate, and ammonium sulfate) revealed that changes in twist resulting from D-cycloserine were always in the opposite direction from those resulting from D-alanine. This antagonism suggests that the biochemical mechanism of twist regulation involves the metabolism of peptidoglycan, particularly reactions involving D-alanine or the dipeptide D-alanyl-D-alanine. This antagonism suggests that the biochemical mechanism of twist regulation involves the metabolism of peptidoglycan, particularly reactions involving D-alanine or the dipeptide D-alanyl-D-alanine. The possibility that peptidoglycan cross-linking is involved is discussed.