SUMMARY: To see whether the initial damage caused by penicillin involved the osmotic barrier, certain characters of Staphylococcus aureus were examined at intervals during the first 2 hr. after addition of penicillin to growing cultures. In the 30 min. following addition of penicillin the cell appeared to expose nearly all its reserve penicillin-binding component (PBC), the penicillin uptake being double that which occurred without growth and the reserve PBC disappearing. The amount of PBC in the cell and its rate of exposure to penicillin in excess of normal synthesis (rate of turnover?) were both very small. Between 30 and 60 min. the uptake of Na, Mg, K, P and increase of total dry matter ceased abruptly. Continued increase in dry wt., while P, Nay Mg and K decreased slightly, resulted in the penicillintreated cells becoming relatively deficient in these elements. Synthesis of lipid phosphorus and some, but not all, large molecule phosphates still continued, and Fe and Co uptake were not affected. After 60-80 min. water entered and solutes began to leave the cell, and synthesis of large molecule P ceased. The primary site of action of penicillin is probably not concerned with gross assimilation of Na, K, Mg, P, Fe, Co, or any substance contributing more than 10% of the dry wt., or with gross synthesis of lipid P. It may, however, involve a reaction turning over 3000 times while the cell mass is doubled.To describe fully the mode of action of a drug one must account for all the changes which take place from the moment of first contact between cell and drug. Although this is clearly impossible to do in detail, some such attempt is necessary as it is very difficult to pin-point any particular defect as being the cause of death. The specific nature of the penicillin molecule and the small amounts bound by cells suggest that the changes caused by the drug are sequences of events branching from perhaps only one small and specific injury. Each event plays its part in a sequence which culminates in the inability of most of the population to divide, and the relative time of appearance of abnormalities is important.It was shown elsewhere (Cooper, 1954) that penicillin is firmly bound in a specific manner by a lipid-containing fraction near the cell wall of a sensitive staphylococcus. Believing that the penicillin-binding component (PBC) of sensitive cells (Rowley, Cooper, Roberts & Lester Smith, 1950) is likely to be the initial site of the bactericidal action of penicillin when acting a t its minimal concentration, it was suggested that its presence in this fraction might indicate that the first effect of penicillin is to disorganize some function specific to the cell wall or to the osmotic barrier. To obtain further information on this possibility, penicillin was added to growing cultures of sensitive staphylococci,