Translocation of membrane-impermeant molecules to the interior of living cells is a necessity for many biochemical investigations. Myristoylation was studied as a means to introduce peptides into living cells. Uptake of a myristoylated, fluorescent peptide was efficient in the B lymphocyte cell line BA/F3. In contrast, this cell line was resistant to uptake of a cell-penetrating peptide derived from the TAT protein. In BA/F3 cells, membrane association was shown to be rapid, reaching a maximum within 30 min. Cellular uptake of the peptide lagged the membrane association but occurred within a similar time frame. Experiments performed at 37 versus 4 degrees C demonstrated profound temperature dependence in the cellular uptake of myristoylated cargo. Myristoylated peptides with either positive or negative charge were shown to load efficiently. In contrast to TAT-conjugated cargo, pyrenebutyrate did not enhance cellular uptake of the myristoylated peptide. The myristoylated peptide did not adversely affect cell viability at concentrations up to 100 muM. This assessment of myristoyl-based transport provides fundamental data needed in understanding the intracellular delivery of myristoylated peptide cargoes for cell-based biochemical studies.