Erythromycin ophthalmic ointment (E. Fougera & Co., Melville, N.Y.) and erythromycin gluceptate standards prepared in ointment base were stored at room temperature and heated at temperatures up to 45°C for as long as 6 h before being assayed for bioactivity. We were unable to detect any significant loss of antibiotic bioactivity.Although the incidence of neonatal gonococcal conjunctivitis has been dramatically reduced by the use of neonatal ocular prophylaxis (6), cases of neonatal gonococcal conjunctivitis (4,5,10) and Chlamydia conjunctivitis (7) still occur. Erythromycin ointment appears to be as effective as silver nitrate in preventing neonatal gonococcal conjunctivitis (2, 16) and does not cause chemical conjunctivitis (14); several reports have therefore recommended its adoption for routine prophylactic use (3, 7, 11). The effects of warming the ointment under the radiant warmer in the delivery room, as is often done before application, or the long-term stability of bioactive erythromycin in such ointment has not been established, although several studies have examined certain other relevant aspects of antibiotic ointments (12,15,17,20). We examined the biological activity of erythromycin in ophthalmic ointment under conditions similar to those used for routine prophylactic administration to neonates for prevention of copjunctivitis and found erythromycin activity to remain stable under all conditions tested.The bioactivity of erythromycin in ointment was determined by using a modified agar diffusion assay, as previously described (1). In brief, pour plates of 1% modified Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) containing 1.5 x 107 CFU of "Micrococcus lysodeikticus" per ml were prepared in polystyrene petri dishes (15 by 150 mm). Wells were produced in the agar with a single cork borer (no. 1 size) with a 3-mm external diameter, the agar plugs were removed by suction, and the erythromycin solutions being tested were placed in the wells (5 pl of solution per well). Each solution was tested in duplicate on two separate bioassay plates (four determinations). After 1 h of refrigeration at 4°C to allow diffusion of the antibiotic, the plates were incubated at 35°C in air for 24 h. Zones of inhibition of bacterial growth surrounding the erythromycin ointment-containing wells were measured using calipers and transmitted light. Zone diameters were fitted to a standard curve of erythromycin concentrations versus diameter derived by using the Cooper equation and methods described by Kavanagh (8). A standard curve (Fig. 1)