When observed in the electron microscope intact gas vesicles appeared as 
transparent areas in whole cells of Microcylus aquaticus, whereas vesicles 
collapsed by centrifugation were not discernible. Within 5 min of suspending 
cells containing collapsed vesicles in growth medium, small transparent vesicles 
were detected. By 15 min the average number of vesicles per cell was 15. This 
number remained relatively constant while the size of the vesicles increased 
until they attained their maximum diamtere of 100 nm. At this time the vesicles, 
interpreted as biconical structures, began to elongate presumably due to the 
synthesis of the cylindrical midsection. Closely correlated with the time at 
which vesicles began to elongate was the initiation of smaller vesicles which 
resulted in a doubling of the number of vesicles per cell by 90 min. This 
evidence coupled with the isolation of a mutant which assembles only the conical 
portions of the vesicle suggests that assembly occurs in two distinct stages 
subject to genetic mutation. Protein and ribonucleic acid synthesis, and 
presumably adenosine triphosphate formation, were required for gas vesicle 
assembly. In addition, inhibition of protein or ribonucleic acid synthesis 
resulted in a loss of extant gas vesicles. Over the time course of our study, 
deoxyribonucleic acid synthesis was not required for gas vesicle assembly or 
stability.