A human head and neck squamous cell carcinoma line (SCC25) derived from a patient with no prior history of radiotherapy or chemotherapy was made resistant to cis-diamminedichloroplatinum(II) (CDDP) by continuous escalation of weekly 30-min pulses of the CDDP from 0 to 0.2 mM over 20 months and then cloned and pulsed weekly with 0.2 mM CDDP for another 20 months. This afforded a resistant subline, SCC25/CP[1], with an IC50 for CDDP 12-fold higher than that of the parental cells. The SCC25/CP[1] cells unexpectedly proved to be cross-resistant to methotrexate (MTX) (24-fold for 30-min treatment and 8-fold for continuous treatment). Resistance was associated with a modest (about 2-fold) increase in the dihydrofolate reductase (DHFR) content according to radioligand-binding assay, and in the rate of cell division. In addition there was a 4-fold decrease in the fraction of long-chain MTX polyglutamates MTX(G4-6) in the cell after 24 h exposure to either 0.2 or 2.0 microM MTX. When the SCC25/CP[1] cells were kept out of CDDP for 8-9 months and 12 months to give the sublines SCC25/CP[2] and SCC25/CP[3], respectively, MTX sensitivity to continuous exposure returned to normal. The SCC25/CP[3] cells still exhibited a slightly elevated DHFR level, but their generation time became shorter than that of the parental SCC25 line. In addition the SCC25/CP[3] cells had an initial uptake velocity (V0) for MTX that was 9-fold greater than the V0 of the SCC25 or SCC25/CP[1] cells, while its ability to form MTX(G4-6) was comparable to that of the SCC25 cells. When SCC25/CP[2] cells were rechallenged with weekly 0.2 mM CDDP pulses for 4-6 months, a MTX-resistant line, SCC25/CP[4], was produced. The SCC25/CP[4] cells retained a slightly elevated DHFR content and a high proliferation rate, but the V0 for MTX influx was intermediate between SCC25 and SCC25/CP[3] cells. The ability to form the longer-chain polyglutamates MTX(G4-6) was again impaired. Thus, MTX cross-resistance can develop in cultured head and neck carcinoma cells when CDDP is used as the selecting agent for primary resistance. MTX resistance is multifactorial, as it is when MTX itself is used as the selecting agent, and appears to involve various combinations of altered growth rate, DHFR content, MTX uptake, and ability to form noneffluxing long-chain MTX polyglutamate species. These results are potentially of clinical relevance, since CDDP and MTX are often used in combination with other drugs or with radiation to treat patients with squamous cell carcinoma of the head and neck.