Cultured skin grafts administered clinically for closure of burn wounds may be contacted by topically applied antimicrobial agents. A study was performed to assess whether commonly used topical antimicrobial agents are toxic to cultured human keratinocytes (HK) in vitro. Serum-free MCDB 153 culture medium containing Neosporin G.U. irrigant (Neomycin, 40 μg/ml-polymyxin B sulfate, 200 units/ml) and a standard tissue culture antimicrobial agent of penicillin (10,000 units/ml)-streptomycin (10,000 μg/ml)-amphotericin B (25 μg/ml) had no effect on the keratinocyte growth rates when compared to standard MCDB 153 medium without antibiotics. Medium containing Sulfamylon (mafenide acetate, 0.85%), Polysporin (polymyxin B sulfate, 1 × 104 units/ml-bacitracin, 500 units/ml), gentamicin sulfate (0.1%), modified Dakins solution (25%), and acetic acid (0.25%) all showed statistically significant (P < 0.01) decreases in keratinocyte growth rates. This data suggests that commonly applied antimicrobials may not be appropriate for cultured grafts in the concentrations that are used clinically. © 1990.